Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

RELATED APPLICATIONS

[0001] The present application claims priority to provisionalapplication U.S. Ser. No. 60/265,151, filed Jan. 31, 2001 (Atty. DocketCL001098-PROV).

FIELD OF THE INVENTION

[0002] The present invention is in the field of kinase proteins that arerelated to the Pftaire kinase subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins, representing two alternative splice forms, thateffect protein phosphorylation and nucleic acid molecules encoding suchpeptide and protein molecules, all of which are useful in thedevelopment of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION Protein Kinases

[0003] Kinases regulate many different cell proliferation,differentiation, and signaling processes by adding phosphate groups toproteins. Uncontrolled signaling has been implicated in a variety ofdisease conditions including inflammation, cancer, arteriosclerosis, andpsoriasis. Reversible protein phosphorylation is the main strategy forcontrolling activities of eukaryotic cells. It is estimated that morethan 1000 of the 10,000 proteins active in a typical mammalian cell arephosphorylated. The high energy phosphate, which drives activation, isgenerally transferred from adenosine triphosphate molecules (ATP) to aparticular protein by protein kinases and removed from that protein byprotein phosphatases. Phosphorylation occurs in response toextracellular signals (hormones, neurotransmitters, growth anddifferentiation factors, etc), cell cycle checkpoints, and environmentalor nutritional stresses and is roughly analogous to turning on amolecular switch. When the switch goes on, the appropriate proteinkinase activates a metabolic enzyme, regulatory protein, receptor,cytoskeletal protein, ion channel or pump, or transcription factor.

[0004] The kinases comprise the largest known protein group, asuperfamily of enzymes with widely varied functions and specificities.They are usually named after their substrate, their regulatorymolecules, or some aspect of a mutant phenotype. With regard tosubstrates, the protein kinases may be roughly divided into two groups;those that phosphorylate tyrosine residues (protein tyrosine kinases,PTK) and those that phosphorylate serine or threonine residues(serine/threonine kinases, STK). A few protein kinases have dualspecificity and phosphorylate threonine and tyrosine residues. Almostall kinases contain a similar 250-300 amino acid catalytic domain. TheN-terminal domain, which contains subdomains I-IV, generally folds intoa two-lobed structure, which binds and orients the ATP (or GTP) donormolecule. The larger C terminal lobe, which contains subdomains VI A-XI,binds the protein substrate and carries out the transfer of the gammaphosphate from ATP to the hydroxyl group of a serine, threonine, ortyrosine residue. Subdomain V spans the two lobes.

[0005] The kinases may be categorized into families by the differentamino acid sequences (generally between 5 and 100 residues) located oneither side of, or inserted into loops of, the kinase domain. Theseadded amino acid sequences allow the regulation of each kinase as itrecognizes and interacts with its target protein. The primary structureof the kinase domains is conserved and can be further subdivided into 11subdomains. Each of the 11 subdomains contains specific residues andmotifs or patterns of amino acids that are characteristic of thatsubdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) TheProtein Kinase Facts Books, Vol 1:7-20 Academic Press, San Diego,Calif.).

[0006] The second messenger dependent protein kinases primarily mediatethe effects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate,cyclic-ADPribose, arachidonic acid, diacylglycerol andcalcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) areimportant members of the STK family. Cyclic-AMP is an intracellularmediator of hormone action in all prokaryotic and animal cells that havebeen studied. Such hormone-induced cellular responses include thyroidhormone secretion, cortisol secretion, progesterone secretion, glycogenbreakdown, bone resorption, and regulation of heart rate and force ofheart muscle contraction. PKA is found in all animal cells and isthought to account for the effects of cyclic-AMP in most of these cells.Altered PKA expression is implicated in a variety of disorders anddiseases including cancer, thyroid disorders, diabetes, atherosclerosis,and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of Internal Medicine, McGraw-Hill, New York, N.Y., pp.416-431, 1887).

[0007] Calcium-calmodulin (CaM) dependent protein kinases are alsomembers of STK family. Calmodulin is a calcium receptor that mediatesmany calcium regulated processes by binding to target proteins inresponse to the binding of calcium. The principle target protein inthese processes is CaM dependent protein kinases. CaM-kinases areinvolved in regulation of smooth muscle contraction (MLC kinase),glycogen breakdown (phosphorylase kinase), and neurotransmission (CaMkinase I and CaM kinase II). CaM kinase I phosphorylates a variety ofsubstrates including the neurotransmitter related proteins synapsin Iand II, the gene transcription regulator, CREB, and the cystic fibrosisconductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBOJournal 14:3679-86). CaM II kinase also phosphorylates synapsin atdifferent sites, and controls the synthesis of catecholamines in thebrain through phosphorylation and activation of tyrosine hydroxylase.Many of the CaM kinases are activated by phosphorylation in addition tobinding to CaM. The kinase may autophosphorylate itself, or bephosphorylated by another kinase as part of a “kinase cascade”.

[0008] Another ligand-activated protein kinase is 5′-AMP-activatedprotein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81).Mammalian AMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in non-lipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

[0009] The mitogen-activated protein kinases (MAP) are also members ofthe STK family. MAP kinases also regulate intracellular signalingpathways. They mediate signal transduction from the cell surface to thenucleus via phosphorylation cascades. Several subgroups have beenidentified, and each manifests different substrate specificities andresponds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R.A. (1993) Nature 365:781-783). MAP kinase signaling pathways are presentin mammalian cells as well as in yeast. The extracellular stimuli thatactivate mammalian pathways include epidermal growth factor (EGF),ultraviolet light, hyperosmolar medium, heat shock, endotoxiclipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-1 (IL-1).

[0010] PRK (proliferation-related kinase) is a serum/cytokine inducibleSTK that is involved in regulation of the cell cycle and cellproliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol.Chem. 271:19402-8). PRK is related to the polo (derived from humans pologene) family of STKs implicated in cell division. PRK is downregulatedin lung tumor tissue and may be a proto-oncogene whose deregulatedexpression in normal tissue leads to oncogenic transformation. AlteredMAP kinase expression is implicated in a variety of disease conditionsincluding cancer, inflammation, immune disorders, and disordersaffecting growth and development.

[0011] The cyclin-dependent protein kinases (CDKs) are another group ofSTKs that control the progression of cells through the cell cycle.Cyclins are small regulatory proteins that act by binding to andactivating CDKs that then trigger various phases of the cell cycle byphosphorylating and activating selected proteins involved in the mitoticprocess. CDKs are unique in that they require multiple inputs to becomeactivated. In addition to the binding of cyclin, CDK activation requiresthe phosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue.

[0012] Protein tyrosine kinases, PTKs, specifically phosphorylatetyrosine residues on their target proteins and may be divided intotransmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs.Transmembrane protein-tyrosine kinases are receptors for most growthfactors. Binding of growth factor to the receptor activates the transferof a phosphate group from ATP to selected tyrosine side chains of thereceptor and other specific proteins. Growth factors (GF) associatedwith receptor PTKs include; epidermal GF, platelet-derived GF,fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF,vascular endothelial GF, and macrophage colony stimulating factor.

[0013] Non-receptor PTKs lack transmembrane regions and, instead, formcomplexes with the intracellular regions of cell surface receptors. Suchreceptors that function through non-receptor PTKs include those forcytokines, hormones (growth hormone and prolactin) and antigen-specificreceptors on T and B lymphocytes.

[0014] Many of these PTKs were first identified as the products ofmutant oncogenes in cancer cells where their activation was no longersubject to normal cellular controls. In fact, about one third of theknown oncogenes encode PTKs, and it is well known that cellulartransformation (oncogenesis) is often accompanied by increased tyrosinephosphorylation activity (Carbonneau H and Tonks NK (1992) Annu. Rev.Cell Biol. 8:463-93). Regulation of PTK activity may therefore be animportant strategy in controlling some types of cancer.

Pftaire Protein Kinases

[0015] The novel human proteins, and encoding gene, provided by thepresent invention are related to Pftaire serine/threonine kinases.Specifically, two alternative splice forms of the same gene are providedby the present invention, referred to herein as “splice form 1” and“splice form 2”. The sequences of a cDNA molecule encoding splice form 1and a transcript sequence encoding splice form 2 are provided in FIG. 1.The amino acid sequences of each splice form are provided in FIG. 2;splice form 1 is 343 amino acids in length and splice form 2 is 435amino acids in length.

[0016] The proteins of the present invention are similar to Pftaire-1previously isolated from the mouse (Besset et al., Mol Reprod Dev 1998May;50(1):18-29 and Lazzaro et al., J Neurochem 1997 Jul;69(1):348-64)and human (Nagase et al., DNA Res 1998 Dec 31 ;5(6):355-64). Pftairekinases are related to Cdk and cdc2 kinases, which are expressed in thebrain and other mitotic tissues; however, Pftaire expression patterns inthe nervous system differ from those of Cdk and cdc2 kinases and Pftairekinases are likely to have distinct functions (Lazzaro et al., JNeurochem 1997 Jul;69(1):348-64).

[0017] Mouse Pftaire-1 shares 50% and 49% amino acid identity with Cdk5and Pctaire-3, respectively. Two transcripts, approximately 5.5 and 4.9kb in size, have been detected. These transcripts are highly expressedin the brain, testis and embryo, and expressed at low levels in allother analyzed tissues in the mouse. Pftaire-1 is expressed in latepachytene spermatocytes in the testis and in post-mitotic neuronal cellsin both the brain and embryo, suggesting that Pftaire-1 plays key rolesin meiosis and neuron differentiation and/or function (Besset et al.,Mol Reprod Dev 1998 May;50(1):18-29).

[0018] Pftaire is highly expressed in both postnatal and adult nervoustissue. Certain terminally differentiated neurons and neuroglia havebeen shown to express Pftaire mRNA and proteins. Pftaire proteins arefound in the nucleus and cytoplasm of neuron cells. These expressionpatterns suggest that Pftaire kinases play key roles in regulating andmaintaining the postmitotic and differentiated condition of nervoussystem cells (Lazzaro et al., J Neurochem 1997 Jul;69(1):348-64).

[0019] Kinase proteins, particularly members of the Pftaire kinasesubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of kinase proteins. The present invention advances the stateof the art by providing previously unidentified human kinase proteinsthat have homology to members of the Pftaire kinase subfamily.

SUMMARY OF THE INVENTION

[0020] The present invention is based in part on the identification ofamino acid sequences of human kinase peptides and proteins, representingtwo alternative splice forms, that are related to the Pftaire kinasesubfamily, as well as allelic variants and other mammalian orthologsthereof. These unique peptide sequences, and nucleic acid sequences thatencode these peptides, can be used as models for the development ofhuman therapeutic targets, aid in the identification of therapeuticproteins, and serve as targets for the development of human therapeuticagents that modulate kinase activity in cells and tissues that expressthe kinase. Experimental data as provided in FIG. 1 indicates expressionin humans in uterus endometrium adenocarcinoma, testis, lungfibroblasts, kidney renal cell adenocarcinoma, and the brain.

DESCRIPTION OF THE FIGURE SHEETS

[0021]FIG. 1 provides the nucleotide sequences of a cDNA molecule (forsplice form 1; SEQ ID NO: 1) and a transcript sequence (for splice form2; SEQ ID NO: 4) that encode the kinase proteins of the presentinvention. In addition, structure and functional information isprovided, such as ATG start, stop and tissue distribution, whereavailable, that allows one to readily determine specific uses of theinventions based on these molecular sequences. Experimental data asprovided in FIG. 1 indicates expression in humans in uterus endometriumadenocarcinoma, testis, lung fibroblasts, kidney renal celladenocarcinoma, and the brain.

[0022]FIG. 2 provides the predicted amino acid sequence of splice form 1(SEQ ID NO: 2) and splice form 2 (SEQ ID NO: 5) of the kinase of thepresent invention. In addition structure and functional information suchas protein family, function, and modification sites is provided whereavailable, allowing one to readily determine specific uses of theinventions based on this molecular sequence.

[0023]FIG. 3 provides genomic sequences that span the gene encoding thekinase proteins of the present invention. (SEQ ID NO: 3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of the inventions based on thismolecular sequence. As illustrated in FIG. 3, SNPs were identified at 26different nucleotide positions (SNPs were also identified at anadditional 30 nucleotide positions 3′ of the ORF, as provided in U.S.Ser. No. 60/265,151, Attrny. Dkt. No. CL1098-PROV, filed Jan. 31, 2001).

DETAILED DESCRIPTION OF THE INVENTION General Description

[0024] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a kinase protein or part of akinase protein and are related to the Pftaire kinase subfamily.Utilizing these sequences, additional genomic sequences were assembledand transcript and/or cDNA sequences were isolated and characterized.Based on this analysis, the present invention provides amino acidsequences of human kinase peptides and proteins, representing twoalternative splice forms (referred to herein as “splice form 1” and“splice form 2”), that are related to the Pftaire kinase subfamily,nucleic acid sequences in the form of transcript sequences, cDNAsequences and/or genomic sequences that encode these kinase peptides andproteins, nucleic acid variation (allelic information), tissuedistribution of expression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thekinase of the present invention.

[0025] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known kinase proteins of thePftaire kinase subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin uterus endometrium adenocarcinoma, testis, lung fibroblasts, kidneyrenal cell adenocarcinoma, and the brain. The art has clearlyestablished the commercial importance of members of this family ofproteins and proteins that have expression patterns similar to that ofthe present gene. Some of the more specific features of the peptides ofthe present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown Pftaire family or subfamily of kinase proteins.

Specific Embodiments Peptide Molecules

[0026] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thekinase family of proteins and are related to the Pftaire kinasesubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the kinase peptides of the present invention, kinase peptides,or peptides/proteins of the present invention.

[0027] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the kinase peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0028] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0029] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0030] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thekinase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0031] The isolated kinase peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in uterus endometrium adenocarcinoma, testis, lung fibroblasts,kidney renal cell adenocarcinoma, and the brain. For example, a nucleicacid molecule encoding the kinase peptide is cloned into an expressionvector, the expression vector introduced into a host cell and theprotein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

[0032] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NOS: 2 and 5),for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NOS: 1 and 4) and the genomicsequences provided in FIG. 3 (SEQ ID NO: 3). The amino acid sequence ofsuch a protein is provided in FIG. 2. A protein consists of an aminoacid sequence when the amino acid sequence is the final amino acidsequence of the protein.

[0033] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ ID NOS:2 and 5), for example, proteins encoded by the transcript/cDNA nucleicacid sequences shown in FIG. 1 (SEQ ID NOS: 1 and 4) and the genomicsequences provided in FIG. 3 (SEQ ID NO: 3). A protein consistsessentially of an amino acid sequence when such an amino acid sequenceis present with only a few additional amino acid residues, for examplefrom about 1 to about 100 or so additional residues, typically from 1 toabout 20 additional residues in the final protein.

[0034] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NOS: 2 and 5), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NOS: 1 and 4) and the genomic sequences providedin FIG. 3 (SEQ ID NO: 3). A protein comprises an amino acid sequencewhen the amino acid sequence is at least part of the final amino acidsequence of the protein. In such a fashion, the protein can be only thepeptide or have additional amino acid molecules, such as amino acidresidues (contiguous encoded sequence) that are naturally associatedwith it or heterologous amino acid residues/peptide sequences. Such aprotein can have a few additional amino acid residues or can compriseseveral hundred or more additional amino acids. The preferred classes ofproteins that are comprised of the kinase peptides of the presentinvention are the naturally occurring mature proteins. A briefdescription of how various types of these proteins can be made/isolatedis provided below.

[0035] The kinase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a kinase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the kinase peptide. “Operatively linked”indicates that the kinase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the kinase peptide.

[0036] In some uses, the fusion protein does not affect the activity ofthe kinase peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant kinase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0037] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the kinase peptide.

[0038] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0039] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the kinase peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0040] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0041] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al,Nucleic Acids Res. 12(l):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4.

[0042] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0043] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the kinase peptides of the present invention as well as beingencoded by the same genetic locus as the kinase peptide provided herein.The gene encoding the novel kinase protein of the present invention islocated on a genome component that has been mapped to human chromosome 2(as indicated in FIG. 3), which is supported by multiple lines ofevidence, such as STS and BAC map data.

[0044] Allelic variants of a kinase peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the kinase peptide as well asbeing encoded by the same genetic locus as the kinase peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. The gene encoding the novel kinase protein of thepresent invention is located on a genome component that has been mappedto human chromosome 2 (as indicated in FIG. 3), which is supported bymultiple lines of evidence, such as STS and BAC map data. As usedherein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a kinase peptide encoding nucleic acid molecule understringent conditions as more fully described below.

[0045]FIG. 3 provides information on SNPs that have been found in thegene encoding the kinase protein of the present invention. SNPs wereidentified at 26 different nucleotide positions (SNPs were alsoidentified at an additional 30 nucleotide positions 3′ of the ORF, asprovided in U.S. Ser. No. 60/265,151 filed Jan. 31, 2001). Some of theseSNPs, which are located outside the ORF and in introns, may affectcontrol/regulatory elements.

[0046] Paralogs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a kinasepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0047] Orthologs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a kinase peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0048] Non-naturally occurring variants of the kinase peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the kinase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a kinase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0049] Variant kinase peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0050] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0051] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as kinase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0052] The present invention further provides fragments of the kinasepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0053] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a kinase peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the kinase peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe kinase peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0054] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inkinase peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0055] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0056] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0057] Accordingly, the kinase peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature kinase peptide is fused withanother compound, such as a compound to increase the half-life of thekinase peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature kinase peptide, such as aleader or secretory sequence or a sequence for purification of themature kinase peptide or a pro-protein sequence.

Protein/Peptide Uses

[0058] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a kinase-effectorprotein interaction or kinase-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0059] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0060] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, kinases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the kinase. Experimental data as providedin FIG. 1 indicates that the kinase proteins of the present inventionare expressed in humans in uterus endometrium adenocarcinoma, testis,lung fibroblasts, and kidney renal cell adenocarcinoma, as indicated byvirtual northern blot analysis. In addition, tissue-screening panelsindicate expression in the brain. A large percentage of pharmaceuticalagents are being developed that modulate the activity of kinaseproteins, particularly members of the Pftaire subfamily (see Backgroundof the Invention). The structural and functional information provided inthe Background and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in uterus endometriumadenocarcinoma, testis, lung fibroblasts, kidney renal celladenocarcinoma, and the brain. Such uses can readily be determined usingthe information provided herein, that which is known in the art, androutine experimentation.

[0061] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to kinases that are related tomembers of the Pftaire subfamily. Such assays involve any of the knownkinase functions or activities or properties useful for diagnosis andtreatment of kinase-related conditions that are specific for thesubfamily of kinases that the one of the present invention belongs to,particularly in cells and tissues that express the kinase. Experimentaldata as provided in FIG. 1 indicates that the kinase proteins of thepresent invention are expressed in humans in uterus endometriumadenocarcinoma, testis, lung fibroblasts, and kidney renal celladenocarcinoma, as indicated by virtual northern blot analysis. Inaddition, tissue-screening panels indicate expression in the brain.

[0062] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the kinase, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, kidney renal cell adenocarcinoma, and thebrain. In an alternate embodiment, cell-based assays involve recombinanthost cells expressing the kinase protein.

[0063] The polypeptides can be used to identify compounds that modulatekinase activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the kinase.Both the kinases of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the kinase. These compounds can befurther screened against a functional kinase to determine the effect ofthe compound on the kinase activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the kinase to a desired degree.

[0064] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the kinase protein and a molecule that normally interacts withthe kinase protein, e.g. a substrate or a component of the signalpathway that the kinase protein normally interacts (for example, anotherkinase). Such assays typically include the steps of combining the kinaseprotein with a candidate compound under conditions that allow the kinaseprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the kinaseprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0065] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L- configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0066] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantkinases or appropriate fragments containing mutations that affect kinasefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0067] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) kinase activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate kinase activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the kinase proteindependent signal cascade can be assayed.

[0068] Any of the biological or biochemical functions mediated by thekinase can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the kinase can be assayed.Experimental data as provided in FIG. 1 indicates that the kinaseproteins of the present invention are expressed in humans in uterusendometrium adenocarcinoma, testis, lung fibroblasts, and kidney renalcell adenocarcinoma, as indicated by virtual northern blot analysis. Inaddition, tissue-screening panels indicate expression in the brain.

[0069] Binding and/or activating compounds can also be screened by usingchimeric kinase proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native kinase. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the kinase is derived.

[0070] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the kinase (e.g. binding partners and/or ligands).Thus, a compound is exposed to a kinase polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble kinase polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble kinase polypeptide, itdecreases the amount of complex formed or activity from the kinasetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the kinase.Thus, the soluble polypeptide that competes with the target kinaseregion is designed to contain peptide sequences corresponding to theregion of interest.

[0071] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the kinase protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0072] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of kinase-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a kinase-binding protein and a candidate compound are incubated inthe kinase protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thekinase protein target molecule, or which are reactive with kinaseprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0073] Agents that modulate one of the kinases of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0074] Modulators of kinase protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the kinase pathway, by treating cells or tissuesthat express the kinase. Experimental data as provided in FIG. 1indicates expression in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, kidney renal cell adenocarcinoma, and thebrain. These methods of treatment include the steps of administering amodulator of kinase activity in a pharmaceutical composition to asubject in need of such treatment, the modulator being identified asdescribed herein.

[0075] In yet another aspect of the invention, the kinase proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent W094/10300), to identify other proteins, whichbind to or interact with the kinase and are involved in kinase activity.Such kinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins or kinase targets as, forexample, downstream elements of a kinase-mediated signaling pathway.Alternatively, such kinase-binding proteins are likely to be kinaseinhibitors.

[0076] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a kinase proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming akinase-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the kinase protein.

[0077] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a kinase-modulating agent, an antisense kinasenucleic acid molecule, a kinase-specific antibody, or a kinase-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0078] The kinase proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, kidney renal cell adenocarcinoma, and thebrain. The method involves contacting a biological sample with acompound capable of interacting with the kinase protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

[0079] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0080] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered kinase activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0081] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0082] The peptides are also useful in pharmacogenomic analysis.Phannacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the kinase protein in which one ormore of the kinase functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and kinase activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0083] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in uterus endometrium adenocarcinoma, testis, lungfibroblasts, kidney renal cell adenocarcinoma, and the brain.Accordingly, methods for treatment include the use of the kinase proteinor fragments.

Antibodies

[0084] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0085] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0086] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0087] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0088] Antibodies are preferably prepared from regions or discretefragments of the kinase proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or kinase/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0089] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0090] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

[0091] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the kinase proteins of the present invention are expressed inhumans in uterus endometrium adenocarcinoma, testis, lung fibroblasts,and kidney renal cell adenocarcinoma, as indicated by virtual northernblot analysis. In addition, tissue-screening panels indicate expressionin the brain. Further, such antibodies can be used to detect protein insitu, in vitro, or in a cell lysate or supernatant in order to evaluatethe abundance and pattern of expression. Also, such antibodies can beused to assess abnormal tissue distribution or abnormal expressionduring development or progression of a biological condition. Antibodydetection of circulating fragments of the full length protein can beused to identify turnover.

[0092] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, kidney renal cell adenocarcinoma, and thebrain. If a disorder is characterized by a specific mutation in theprotein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

[0093] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin uterus endometrium adenocarcinoma, testis, lung fibroblasts, kidneyrenal cell adenocarcinoma, and the brain. The diagnostic uses can beapplied, not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

[0094] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0095] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in uterusendometrium adenocarcinoma, testis, lung fibroblasts, kidney renal celladenocarcinoma, and the brain. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

[0096] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the kinase peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0097] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

Nucleic Acid Molecules

[0098] The present invention further provides isolated nucleic acidmolecules that encode a kinase peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the kinase peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0099] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0100] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0101] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0102] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NOS: 1 and 4, transcript sequence and SEQ ID NO: 3, genomicsequence), or any nucleic acid molecule that encodes the proteinprovided in FIG. 2, SEQ ID NOS: 2 and 5. A nucleic acid moleculeconsists of a nucleotide sequence when the nucleotide sequence is thecomplete nucleotide sequence of the nucleic acid molecule.

[0103] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NOS: 1 and 4, transcript sequence and SEQ ID NO: 3, genomicsequence), or any nucleic acid molecule that encodes the proteinprovided in FIG. 2, SEQ ID NOS: 2 and 5. A nucleic acid moleculeconsists essentially of a nucleotide sequence when such a nucleotidesequence is present with only a few additional nucleic acid residues inthe final nucleic acid molecule.

[0104] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NOS:1 and 4, transcript sequence and SEQ ID NO: 3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NOS: 2 and 5. A nucleic acid molecule comprises a nucleotide sequencewhen the nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0105] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0106] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0107] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the kinase peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0108] Isolated nucleic acid molecules can be in the form of RNA, suchas MRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0109] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the kinase proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0110] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0111] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or MRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0112] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0113] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel kinase protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 2 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data.

[0114]FIG. 3 provides information on SNPs that have been found in thegene encoding the kinase protein of the present invention. SNPs wereidentified at 26 different nucleotide positions (SNPs were alsoidentified at an additional 30 nucleotide positions 3′ of the ORF, asprovided in U.S. Ser. No. 60/265,151 filed Jan. 31, 2001). Some of theseSNPs, which are located outside the ORF and in introns, may affectcontrol/regulatory elements.

[0115] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6X sodiumchloride/sodium citrate (SSC) at about 45C., followed by one or morewashes in 0.2 X SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

[0116] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs were identified at 26 differentnucleotide positions (SNPs were also identified at an additional 30nucleotide positions 3′ of the ORF, as provided in U.S. Ser. No.60/265,151, Attny. Dkt. No. CL1098-PROV, filed Jan. 31, 2001).

[0117] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0118] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0119] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0120] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0121] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. The gene encoding the novelkinase protein of the present invention is located on a genome componentthat has been mapped to human chromosome 2 (as indicated in FIG. 3),which is supported by multiple lines of evidence, such as STS and BACmap data.

[0122] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0123] The nucleic acid molecules are also useful for designingribozymes corresponding to 25 all, or a part, of the mRNA produced fromthe nucleic acid molecules described herein.

[0124] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0125] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0126] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0127] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the kinase proteins of the present invention areexpressed in humans in uterus endometrium adenocarcinoma, testis, lungfibroblasts, and kidney renal cell adenocarcinoma, as indicated byvirtual northern blot analysis. In addition, tissue-screening panelsindicate expression in the brain. Accordingly, the probes can be used todetect the presence of, or to determine levels of, a specific nucleicacid molecule in cells, tissues, and in organisms. The nucleic acidwhose level is determined can be DNA or RNA. Accordingly, probescorresponding to the peptides described herein can be used to assessexpression and/or gene copy number in a given cell, tissue, or organism.These uses are relevant for diagnosis of disorders involving an increaseor decrease in kinase protein expression relative to normal results.

[0128] In vitro techniques for detection of MRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0129] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a kinase protein, such as bymeasuring a level of a kinase-encoding nucleic acid in a sample of cellsfrom a subject e.g., MRNA or genomic DNA, or determining if a kinasegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat the kinase proteins of the present invention are expressed inhumans in uterus endometrium adenocarcinoma, testis, lung fibroblasts,and kidney renal cell adenocarcinoma, as indicated by virtual northernblot analysis. In addition, tissue-screening panels indicate expressionin the brain.

[0130] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate kinase nucleic acid expression.

[0131] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the kinase gene, particularly biological and pathologicalprocesses that are mediated by the kinase in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin humans in uterus endometrium adenocarcinoma, testis, lungfibroblasts, kidney renal cell adenocarcinoma, and the brain. The methodtypically includes assaying the ability of the compound to modulate theexpression of the kinase nucleic acid and thus identifying a compoundthat can be used to treat a disorder characterized by undesired kinasenucleic acid expression. The assays can be performed in cell-based andcell-free systems. Cell-based assays include cells naturally expressingthe kinase nucleic acid or recombinant cells genetically engineered toexpress specific nucleic acid sequences.

[0132] The assay for kinase nucleic acid expression can involve directassay of nucleic acid levels, such as MRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the kinase proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0133] Thus, modulators of kinase gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of kinase MRNA inthe presence of the candidate compound is compared to the level ofexpression of kinase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of MRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0134] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate kinase nucleic acid expressionin cells and tissues that express the kinase. Experimental data asprovided in FIG. 1 indicates that the kinase proteins of the presentinvention are expressed in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, and kidney renal cell adenocarcinoma, asindicated by virtual northern blot analysis. In addition,tissue-screening panels indicate expression in the brain. Modulationincludes both up-regulation (i.e. activation or agonization) ordown-regulation (suppression or antagonization) or nucleic acidexpression.

[0135] Alternatively, a modulator for kinase nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thekinase nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inhumans in uterus endometrium adenocarcinoma, testis, lung fibroblasts,kidney renal cell adenocarcinoma, and the brain.

[0136] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe kinase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0137] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in kinase nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in kinase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the kinase gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the kinase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a kinase protein.

[0138] Individuals carrying mutations in the kinase gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the kinaseprotein of the present invention. SNPs were identified at 26 differentnucleotide positions (SNPs were also identified at an additional 30nucleotide positions 3′ of the ORF, as provided in U.S. Ser. No.60/265,151 filed Jan.31, 2001). Some of these SNPs, which are locatedoutside the ORF and in introns, may affect control/regulatory elements.The gene encoding the novel kinase protein of the present invention islocated on a genome component that has been mapped to human chromosome 2(as indicated in FIG. 3), which is supported by multiple lines ofevidence, such as STS and BAC map data. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way. In some uses, detection of the mutationinvolves the use of a probe/primer in a polymerase chain reaction (PCR)(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa etal., PNAS 91:360-364 (1994)), the latter of which can be particularlyuseful for detecting point mutations in the gene (see Abravaya et al.,Nucleic Acids Res. 23:675-682 (1995)). This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0139] Alternatively, mutations in a kinase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0140] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0141] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant kinase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0142] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 21 7:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0143] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the kinase gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding the kinaseprotein of the present invention. SNPs were identified at 26 differentnucleotide positions (SNPs were also identified at an additional 30nucleotide positions 3′ of the ORF, as provided in U.S. Ser. No.60/265,151 filed Jan. 31, 2001). Some of these SNPs, which are locatedoutside the ORF and in introns, may affect control/regulatory elements.

[0144] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0145] The nucleic acid molecules are thus useful as antisenseconstructs to control kinase gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of kinase protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into kinase protein.

[0146] Alternatively, a class of antisense molecules can be used toinactivate MRNA in order to decrease expression of kinase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired kinase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the MRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the kinase protein, such as substratebinding.

[0147] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in kinase geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired kinaseprotein to treat the individual.

[0148] The invention also encompasses kits for detecting the presence ofa kinase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the kinase proteins of the presentinvention are expressed in humans in uterus endometrium adenocarcinoma,testis, lung fibroblasts, and kidney renal cell adenocarcinoma, asindicated by virtual northern blot analysis. In addition,tissue-screening panels indicate expression in the brain. For example,the kit can comprise reagents such as a labeled or labelable nucleicacid or agent capable of detecting kinase nucleic acid in a biologicalsample; means for determining the amount of kinase nucleic acid in thesample; and means for comparing the amount of kinase nucleic acid in thesample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect kinase protein mRNA or DNA.

Nucleic Acid Arrays

[0149] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1, 3, and 4).

[0150] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Cheeet al), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0151] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0152] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0153] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationW095/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0154] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0155] Using such arrays, the present invention provides methods toidentify the expression of the kinase proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the kinasegene of the present invention. FIG. 3 provides information on SNPs thathave been found in the gene encoding the kinase protein of the presentinvention. SNPs were identified at 26 different nucleotide positions(SNPs were also identified at an additional 30 nucleotide positions 3′of the ORF, as provided in U.S. Ser. No. 60/265,151 filed Jan. 31,2001). Some of these SNPs, which are located outside the ORF and inintrons, may affect control/regulatory elements.

[0156] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1 982), Vol.2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0157] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0158] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0159] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0160] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified kinase gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

Vectors/Host Cells

[0161] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0162] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0163] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0164] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0165] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0166] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0167] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0168] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0169] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0170] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0171] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0172] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterokinase. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharrnacia, Piscataway,N.J.) which fuse glutathione S-transferase (GST), maltose E bindingprotein, or protein A, respectively, to the target recombinant protein.Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185:60-89(1990)).

[0173] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0174] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J6:229-234 (1987)), pMFa (Kurjan et al, Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0175] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0176] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J 6:187-195 (1987)).

[0177] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0178] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0179] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0180] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0181] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0182] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0183] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0184] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell- free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0185] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such askinases, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0186] Where the peptide is not secreted into the medium, which istypically the case with kinases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0187] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

Uses of Vectors and Host Cells

[0188] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga kinase protein or peptide that can be further purified to producedesired amounts of kinase protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0189] Host cells are also useful for conducting cell-based assaysinvolving the kinase protein or kinase protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native kinase protein is useful forassaying compounds that stimulate or inhibit kinase protein function.

[0190] Host cells are also useful for identifying kinase protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantkinase protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native kinase protein.

[0191] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a kinase proteinand identifying and evaluating modulators of kinase protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0192] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the kinase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0193] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the kinase protein to particularcells.

[0194] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0195] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0196] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0197] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, kinase protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo kinaseprotein function, including substrate interaction, the effect ofspecific mutant kinase proteins on kinase protein function and substrateinteraction, and the effect of chimeric kinase proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more kinase proteinfunctions.

[0198] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 10 1 2203 DNA Human 1 gtgagtcata tgaaagctcc acgctgctga cctctggcaaaaagggagag aacaaggata 60 ggagaggcag tgggggaaag gttcaagtgc gggttttctccttgaaccta gaagattatg 120 ggtcaagagc tgtgtgcaaa gactgtacag cctggatgcagctgctacca ttgttcagag 180 ggaggcgagg cacacagctg tcggaggagt cagcctgagaccacggaggc tgcgttcaag 240 ctaacagacc taaaagaagc atcatgttcc atgacttcatttcaccccag gggacttcaa 300 gctgcccgtg cccagaagtt caagagtaaa aggccacggagtaacagtga ttgttttcag 360 gaagaggatc tgaggcaggg ttttcagtgg aggaagagcctcccttttgg ggcagcctca 420 tcttacttga acttggagaa gctgggtgaa ggctcttatgcgacagttta caaggggatt 480 agcagaataa atggacaact agtggcttta aaagtcatcagcatgaatgc agaggaagga 540 gtcccattta cagctatccg agaagcttct ctcctgaagggtttgaaaca tgccaatatt 600 gtgctcctgc atgacataat ccacaccaaa gagacactgacattcgtttt tgaatacatg 660 cacacagacc tggcccagta tatgtctcag catccaggagggcttcatcc tcataatgtc 720 agacttttca tgtttcaact tttgcggggc ctggcgtacatccaccacca acacgttctt 780 cacagggacc tgaaacctca gaacttactc atcagtcacctgggagagct caaactggct 840 gattttggtc ttgcccgggc caagtccatt cccagccagacatactcttc agaagtcgtg 900 accctctggt accggccccc tgatgctttg ctgggagccactgaatattc ctctgagctg 960 gacatatggg gtgcaggctg catctttatt gaaatgttccagggtcaacc tttgtttcct 1020 ggggtttcca acatccttga acagctggag aaaatctgggaggtgctggg agtccctaca 1080 gaggatactt ggccgggagt ctccaagcta cctaactacaatccaggtaa tattgatctg 1140 agcttttgaa tactctgaga attagtaatg taaggagagcattggccacg ctaacagggc 1200 gttcttgtat tgtgaactca gcggcaaaga tgggtgtagaggaatttcta cattcatata 1260 ttccctgact aatctttgta tgaggaagac actgaaagagtagctgaggt tagaccagtt 1320 ccccagctct gtaaaacaca agtagcaagc tgaatagaatttgaaatgac tattactgtg 1380 gattccacat ccattgtcaa atacccaatg gctcaaaagaacaactcaaa agatgggctc 1440 acttttgggc cccctgactg tcataagtgt attgattagtattgaattgc atatgtataa 1500 aaagaaagct aatgcaacag aacagaggta gaggctcgctaggcctagga catgccaagt 1560 aagctgtctg taggttatac ttactaagag ttcattcattgcctgtaaac ctgacacttg 1620 gtcattgtct ctcacacatt tcatctttca agactggcttctgggatcga tttagaagtg 1680 ctggaagtgt tatccatggg ggaattcttt gagaagctgtcgcagggcca catcagaggg 1740 atcagattaa gcagtagtca cttcaaggat gttgagacagaggggaggag acaggcactg 1800 aactacagga tgaaggatca tattagaagc tgaagaagcaaataaagccc atgccaaagc 1860 tgagctctca ctggcagggt tgaaggggag gtagaaaggtacagataacg acaagattag 1920 ggtggatatg ctccaagcca gatttttcta gtctttatggtcttacattg ttccattact 1980 aaaaatgaaa tcttcccaaa ttgttgtcct tacttttttttttttttttt gagatggagt 2040 tttgctctta tcgcccaggc tggagtgcag tgagccgagattgcgccact gcatgtccgc 2100 agtccgacct gggcgacaga gcgagactcc gtctcaaaactaaaaaaaaa aaaaaaaaaa 2160 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaa 2203 2 343 PRT Human 2 Met Gly Gln Glu Leu Cys Ala Lys Thr Val GlnPro Gly Cys Ser Cys 1 5 10 15 Tyr His Cys Ser Glu Gly Gly Glu Ala HisSer Cys Arg Arg Ser Gln 20 25 30 Pro Glu Thr Thr Glu Ala Ala Phe Lys LeuThr Asp Leu Lys Glu Ala 35 40 45 Ser Cys Ser Met Thr Ser Phe His Pro ArgGly Leu Gln Ala Ala Arg 50 55 60 Ala Gln Lys Phe Lys Ser Lys Arg Pro ArgSer Asn Ser Asp Cys Phe 65 70 75 80 Gln Glu Glu Asp Leu Arg Gln Gly PheGln Trp Arg Lys Ser Leu Pro 85 90 95 Phe Gly Ala Ala Ser Ser Tyr Leu AsnLeu Glu Lys Leu Gly Glu Gly 100 105 110 Ser Tyr Ala Thr Val Tyr Lys GlyIle Ser Arg Ile Asn Gly Gln Leu 115 120 125 Val Ala Leu Lys Val Ile SerMet Asn Ala Glu Glu Gly Val Pro Phe 130 135 140 Thr Ala Ile Arg Glu AlaSer Leu Leu Lys Gly Leu Lys His Ala Asn 145 150 155 160 Ile Val Leu LeuHis Asp Ile Ile His Thr Lys Glu Thr Leu Thr Phe 165 170 175 Val Phe GluTyr Met His Thr Asp Leu Ala Gln Tyr Met Ser Gln His 180 185 190 Pro GlyGly Leu His Pro His Asn Val Arg Leu Phe Met Phe Gln Leu 195 200 205 LeuArg Gly Leu Ala Tyr Ile His His Gln His Val Leu His Arg Asp 210 215 220Leu Lys Pro Gln Asn Leu Leu Ile Ser His Leu Gly Glu Leu Lys Leu 225 230235 240 Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Ile Pro Ser Gln Thr Tyr245 250 255 Ser Ser Glu Val Val Thr Leu Trp Tyr Arg Pro Pro Asp Ala LeuLeu 260 265 270 Gly Ala Thr Glu Tyr Ser Ser Glu Leu Asp Ile Trp Gly AlaGly Cys 275 280 285 Ile Phe Ile Glu Met Phe Gln Gly Gln Pro Leu Phe ProGly Val Ser 290 295 300 Asn Ile Leu Glu Gln Leu Glu Lys Ile Trp Glu ValLeu Gly Val Pro 305 310 315 320 Thr Glu Asp Thr Trp Pro Gly Val Ser LysLeu Pro Asn Tyr Asn Pro 325 330 335 Gly Asn Ile Asp Leu Ser Phe 340 353332 DNA Human 3 tataggccaa tgctgtggct cacgcgtgta ttcccagcac tttgggaggcaggaggatcg 60 cttgagctca ggaattggag acaagcctac gtaacatagt gaaacctctgtctgtacaaa 120 taataaaaga attttccagg catggtggcg tgcaccccca gtgccagctatttgggaggc 180 tgaggtagga ggaatgcttg aagccaggag ttgaagacaa gcctaggcaacatagtgaga 240 ccctgtgtct ataaaaaata attagctggt tgtcttggca caggcctgcagctagctact 300 cggaagactg aggtgggagg atcactgagc ccaggaggct gaggctgcagtgaacagtga 360 tcacccagct ggattccagc ctggaagaca gagggagacc ctgtttccaaaaaaaaaaaa 420 aaaaaaaaat gcaagaaaag acatcataaa cttgacctgg gacataacttttatgtgatg 480 aaattcacaa tcttttagga agaaattagc atttctgata aaatgtattataattatatt 540 attataaatt caaatggaat taaatattct gagaaactag cttctcactctctcagttgt 600 cagtcaaaac tttaatggtc tttggccggg tgcggtggct cacgcctgtaatcccagcac 660 tttgggaggc cgaggcgggt ggatcacaag gttaggagat cgagaccatcctggctaaca 720 cggtgaaacc tcgtctctac taaaaataca aaaaattagc cgggtgcggtgccagacgcc 780 tgtagtccca gctgctcagg aggctgaggc aggagaatgg tgtgaacccgggaggcggag 840 cttgcagtga gccgagattg cgccactgca ctccagcctg ggcgacagtgcgagactctg 900 tctcaaaaaa aaaaaaaaaa aaaagttgaa tggtctttga gccaagtagtcttccttttc 960 ttcttcttct tttttttttt ttttcaaaaa atatctctag attgaatcttggaattggct 1020 taagtctctt ctcttgtggc aattttgaaa tgaaaaaata catgctcataattaaattac 1080 ctgaacattt taaaaaacca tcatgaggtt caaatatcaa atattcataaatattgttgt 1140 gataatagac ataactctta ttttttccct taataatgat tgtttatatatcctccattc 1200 tgtctcactt tatgattagt atattatagt ggcaataatc ttaggaatctaacagagaaa 1260 agtgttgcat ttgaagacta cagactgcaa accaatttaa gccagattccttgacatgtt 1320 gtgctgttaa tatagtactt tacatatagt aaacattaat tacatatatgtggaaggaag 1380 caagcaagaa aggaagaaag tatttcattc aaactcctct ctctccatcaccattggcta 1440 atatcatcat ttgtacagtt aagaacaaca taggtgctca ccacatagtttttgaataaa 1500 tgaatgaatg gcaacccttc taagactatt ggatacacta ttgtttgaaggcaaagagat 1560 gcagtagata ttttcaactt ttttcctgtt ttatgattct gtggtttctttgactactaa 1620 aagttagcta ggtagcaaat ttgttttaaa gtctgaaaac caaaatgctttcagataaaa 1680 ggtagggaga aaaatactcc tcaacatgtc cactttagca ccaggaaaacctaatatcaa 1740 tatcaccatc aatgatatca tataaatatc attgcataga taagcaatgtcaatccctaa 1800 aaactatgta taccaatagc actaacttgt ggccagaaca agaaccttaactgtgccaaa 1860 ttttattcta ttcaataaca gctgcctcgt tttcagttgt gcacatctgaatgcaagcaa 1920 tccctgtctg atgtggagtt tcttgcactg ataaggaaaa actgctgaagttgtgaggct 1980 gctccaggca gagccatcat gtgagtcata tgaaagctcc acgctgctgacctctggcaa 2040 aaagggagag aacaaggata ggagaggcag tgggggaaag gttcaagtgcgggttttctc 2100 cttgaaccta caagattatg ggtcaagagc tgtgtgcaaa gactgtacagcctggatgca 2160 gctgctacca ttgttcagag ggaggcgagg cacacagctg tcggaggagtcagcctgaga 2220 ccacggaggc tgcgttcaag gtatttgtat cccaggagag agcatctttctctattgata 2280 aaccaaggag ttcagacact ccctttttgt agcgggatct gattcttctgcggtaggtct 2340 aaaccaataa aatgaaaatt ctattaaagt cacagaaaat ttatggctgtagttatcaaa 2400 tttggggaat ttcttgtaaa ccaaaaggga aaaataatcc ttggctttgggctgcacgaa 2460 actcacttgg cttgaagtcg agaaagtagt tctctcaaaa tctctaaggtcctaaattac 2520 agagctgaaa cttaaaaggc aagctgcagt attagttggt atgctatggatttgaaactt 2580 tagtaattag ttcatgatta ttagcaatgc catagattat tcccctacagcaataaatta 2640 agtggacatg aaaaaaaaaa gccagactta aacagaaaaa agttgcaaaacatccatcaa 2700 agagatttag gttaacctga atgttaaaga cacattttta ggtgaagaaagaatgtagta 2760 tttcaggagt tgataccatt atggtctttt tcagggatct ttcaagaaaagtgccttttg 2820 ggggtacagg aagcttagaa aacatttgaa gagtgaaaat gaggcaaataaagaaaaaat 2880 ggttttacca ggcactgaat ctttactttg cataaatttt atttctgctctttctttttt 2940 ctctagctaa cagacctaaa agaagcatca tgttccatga cttcatttcaccccagggga 3000 cttcaagctg cccgtgccca gaagttcaag agtaaaaggc cacggagtaacagtgattgt 3060 tttcaggaag aggatctgag gcagggtttt cagtgggtga gtgagcagctgatgttgatc 3120 aagaagaatt taatgtgagc ttgtctacgg aggccggccc ttgcttccagggcaattact 3180 gagcgagcct tcccaagtct gctctggcaa tgctgtctaa tttccctggggaaaaaaagt 3240 caacactaaa aaaaagtgtt ctttctctct tccctttcac ccgctccttttccccattcc 3300 cctagagcag aggaagagcc tcccttttgg ggcagcctca tcttacttgaacttggagaa 3360 gctgggtgaa ggctcttatg cgacagttta caaggggatt agcaggtgagtgacacatag 3420 ctgggagaga ctttagagat gagagtcccg cccccccaat ttcatattataaagccaggt 3480 gagacatcat agaagttcat agcactcagg acctgtgcaa gacaccatggccgacaggga 3540 gagagacatg ataacttaaa cagccttgaa agaaaaacaa acctgccctgccctaattaa 3600 aatcagccca cttaaatgtt tatcagcctt tcccttcttg cattcaattcagagaattca 3660 aagaaaatag acattctcta ctactgaccc aaagaacaat tatcactcttcaggcctgtg 3720 ggaggcacag ttggtaaagc gtctctaaca ggttttttat atccctccctaaatcacaat 3780 gacagagttt tgtaatggca acctggaatt tgctgcttca ttcctccacctggcctttat 3840 agaagaaact gaagttggtt tctgcaaatt atggtacatg caaaagatgataaatcctag 3900 attttttata tttgcaaaat acacaaaatg tctggagaat aaaaatactgcttatccaaa 3960 agctaagtac taattttggt aaacaaccaa ctttgttaaa tatatgtaaaagatccatga 4020 attccccttt tagtcaaggt gggaaagttg gatggtcgct tttttctttatgttactcca 4080 atagagagaa aagtaatggc tcaatagtgg ttaaatatta attttaaaaatatagctgat 4140 ccgagtgcag tggtgtttac aactacttga tcacaaccag ttacagatttctttgttcct 4200 tctccactcc cactgcttca cttaactggc caaaaacgaa aaaagaaaaattttatataa 4260 ctactacaag actaaatatt tattatttat cttagtattt atgctgttattattattttt 4320 acttgttaaa acaggattgt aggggacata cagttttatt ttattttattatttatatat 4380 ttatttattt attttggaat ggaatctctg tcacccacgc tggagtgcagtggtgcgatc 4440 tcagatgact gcaacctctg cctcctgagt tcaagcaact ctcctgcccctggcccttta 4500 tactttctta atctgtttta gtcatggtgt accttaactt ttttcaatgctgagaacatc 4560 tgcaataaag gaccacattt tattttattc taagcttcct catatcaatttggccatggt 4620 aactgttttc aaggtggctc ggaacggggg caccctggaa catacttggatacatgggca 4680 ccatggacac ttctgatcct ctcttctgag ttctgacttt gattgttctgcacagacctt 4740 tccagcccga agtttacaca gaattcactt atcttttctt ctagttactttatgttttct 4800 ttttcattta actctttcat ctactgggaa tttatattgt atattcacaatcaccccagc 4860 tccatttatt agattttctt ttctctgatg gtttgaaatg ctgccatgattatatattag 4920 atctcacgaa tacttgaaat tctttctgtt ctaatctttt aaaaatcatgtttccttaat 4980 ctatcttttc ttatatttgt gctgcatgat tttaattatt gttgctttaggctattttta 5040 gaatatatca aaactctacg ttagagaatt attgacatct ttgcattattagattttcta 5100 atacaaatat cctgtaaata tctaatacaa cagtctctgg atggtcactgtacaagaccc 5160 tatagaatcc ctaccctcca ttccccggca cacactcagc tcctccctgtcctcatctcc 5220 ttcccctctc ctgcttcaat gacagactgc tcctgcctca gtcaaggacttttaacttgc 5280 tgttccctct gcctggagct gccttccact gttcatgcac acagctgactccccctcgcc 5340 atcagattcc tggttcaagt gttaccttat ttataaaact gtagtcccagctagtccagg 5400 gaggctggag gcaggagaat cacttgaact ttggaggcag aggttgcagtgagctgagat 5460 cggcaccacc gcactccagc ctgggtgaga gtgacactgt ctcaaaaaaaaaaaaaagca 5520 ttttctctta taaacatatt tgccaaaaaa ctttttgcag ggtttgggggagaatttcac 5580 agaaccatgt tctgaggaaa atacttacct cataaaactc taaaacaaaatttcaaagac 5640 atgataaggc aaacaaaaga aactggggaa aagtatatgc aaaatagttcaataaaaagg 5700 tgggcaaatc ggcaaatcac aagaaaaaca gaaaagatcc ataaacttatgaaaagtcag 5760 tttcacatat ggttaaagaa atataaatta aaatgcgata aacctttttacttttcaaat 5820 aggccaaaaa aaaaaagaag atgaaagcga aaagccaacc cacatgatagggctatgaca 5880 gagggacaca ggagccaact gaaagagctt ccaaaggaca aagctgcaaaaatatgagca 5940 accaaaaaaa gtggtattaa attataaccc aaagtataaa ataaatatctatgagtccgt 6000 actgatataa ataaatgatt caatacatta acaaatggga gagaagaaacaaatctctca 6060 tgccaaataa atacaaataa tttatgtaga taatatacct tcaaagaggtacagcataac 6120 tctccactcc ttaagtgtgg gtcattcata gtggcatttc tctaaaagtacagtatgaaa 6180 aagggggaga aagagtaact ttagagtaga gaaacctgac caacactatctcagacaggt 6240 gactaaggtc aacatcaaaa gtcataaatc atgatgatgg tatgcactcttttttttttt 6300 tttttttttt ttctcagatg gagtctcact ctgtcgccca ggctggggtgcagtggcgca 6360 atctcagctc actgcaacct ccggctcccg ggttcaagcg attctcctctcagcctcctg 6420 agtagctggg atcacaggcg cgtgccacca tacccggcta attttttgtattttagtaga 6480 gacggggttt caccatgttg cccaggctgg tctcaaactc ccgagctcaggcaatccacc 6540 cacctcaacc tcccaaagtg ctaggattac aggcatgagc cactgcgcctggctgagggt 6600 atgcactttt tttttttttg agacggagtc ttgctctgtc gcccaggctggagtgcagtg 6660 gcacgatctt ggctcactgc aagctccgcc tcccaggttc acgccattctcctgcctcag 6720 cctccccagt agctgggact acaaggtgcc ccaccaccca cacccggctaattttttgta 6780 tttttagtag agacggggtt tcactgtgtt aggcaggatg gtctcgatctcctgacctcc 6840 tgatccaccg gccttcgcct cccaaagtgc tgggattaca ggcgtgagccactgtgcccg 6900 gcctgatgaa atgttaaatc tttattaaat atcggattgt acaagaatgaactataagag 6960 aaaagttaca tggaggaaaa aaggttacta acaatatgat tttaatcccactgtattaaa 7020 aacaatggat ttatacctgc attaaaatct tctctattct cagcacttagctgatatgaa 7080 taaaatgatg aatgagggga cagtaggagg aaatgaagag agagagaataatggtgtggc 7140 ctgggaagat caggtagcac ttagaagccc gctgcaagaa tttggcttttattctaagta 7200 atgcgtggag atatggtggc ttttgaacag aaaagtgact tgtcctgattgtcatttgaa 7260 aagtatgcct ccaactacta ctgctgagag taaatagtag gagtgcaagtgtgctcagca 7320 gggaaactgt tagaagacca ctacaaggct gggcttggtg gctcgtgcctgtaatcccag 7380 cactttggga gcctgacgtg ggcagatcac ctgaggtcag gagttcgagaccagcctggc 7440 caaaatggtg aaacccccat ctctgctaaa aatacaaaaa ttagccaggtgtggtggggg 7500 tcccctgtaa tcccagcttc ttgggaggct gaggcaggag aattgcttgaacccaggagg 7560 tggaggttgc agtgagccaa gatcgtgcca ctgtactcca gcctgggcaacagagcgaga 7620 ttctgtctca aaaaaaaaaa aaaaaaacaa aaaaacaaaa aaacactacaataagtcaga 7680 tgaaaaataa taataagctc caaattttct ataatggaca tatatatatatatcacttta 7740 gtaaagaggg aaaatgcttt ggaatatata tgttatatat gtattgatacatgttaaact 7800 ttttattttg agaaaattat agatttatat gctagaatat attttgaagtgaaagtgctt 7860 ttgttaagcc atctttggta taaattgctg ctttgaacca cctcaataagtgtgtgcccc 7920 tcaatccctc tcttctagaa taaatggaca actagtggct ttaaaagtcatcagcatgaa 7980 tgcagaggaa ggagtcccat ttacagctat ccgagaaggt aagaacagcagaaatggacc 8040 caatagatct gttttgagtc cttgatttgg taaaaaatgt attgcattgatccattcagc 8100 atctagtttt gattcttctg gaatactata attacatttt tatttttcatacaagttttt 8160 caagaaattt acactgctat tttattactt aattttgagg aaattgagatttaaaactat 8220 tatatcactt gaccaaaact ataaattcac tgagcaatta ctaatactttccatgtgttt 8280 ggcctcatgc taggtgctaa ggctatacct atataacctc agaaaattcctataaaagag 8340 aaaatatata atcacacaaa ttcttactgg gaaatttgcc tgaacataacatgttgttag 8400 ctagcacttg gagattctcc agaaggcatg catgtttagt gttactgcctgtattttctc 8460 tgtgccctgg acagtacagc aaatgggtga ggaacctggt gtcaaatggacttgggtttg 8520 cagcacaggt ccaccaatca ctagtggtat gatgttgggt aggttactttagctatttat 8580 tactcagttt cttgcaggaa gaggataata gtggtaccta tttcatggagttgttatgag 8640 tattcaacaa gaatatgtat ataaagcact tatcacagag tcagtttttcagagttcaac 8700 aaatgttgac catttttatt ccattcttct tttcctgggt aatgtcttatttaccatcaa 8760 gataactaat actttataac ataaacatca agaagccaac atagtgaaatgaatcattaa 8820 aaatataatt tatcaacctt tattgcatga gccatttgaa ataagatgatgataggattg 8880 ctatgcattt cagcaaaatc ccagagaaat ggcacttccc tggccttattttctcccact 8940 tttaactact tatcttctgt tctttactga gcacatgcta tatgcagagtatgctgctgg 9000 atgctgtgaa ggatgagaag agaaacccat gtctttgttc tatcatttgcagtcttaaca 9060 gagcacatga ttcaagttac aagtgtataa aagacataaa ctaagatgagagcaagttag 9120 tctcagtgtg actgatggag tcactagatt ttgaactgag cttggaaggataggttatgc 9180 aaacaagcat ggaaaaagca attcagaaaa tgagtttata actgaatttgataccctttt 9240 caaaagtctt tcagagcccc tgaggaatac atcattttga atttaattggaagggccaaa 9300 tgggctattg gtttagccag agattcatcc tggtaggatc aggtgcattctgggagaagg 9360 catggtttta agtgtttaat ataatggaaa ctgcattaac taatgtacttattaatggtc 9420 tccatgaaag gatgatcaga tttggaaaga gatgtatgga taggttaaagagtatttgtg 9480 aacgtaatag aaattcccag gtcacccgca taagaggaag gtttcctttgtgagcttgag 9540 tttgccaatt gcttaagatt ggctttgctt agatattgcc cacagccaagtttttcaggt 9600 tgacatttaa ctgtaacagt gaaacctttt gccaggtttg ctaacagatggttctcagca 9660 tggttcagaa aacctggatc cgttttcttc tgtatgctaa atgtttctttcattgcatat 9720 ttacggagga attgcctctc catcacaggt gtttacaatt acatttagtagtcaactgtg 9780 gactttcttg gtttgtttta tggacttacc ttaccgaatg ctttgctcgtgtaatattaa 9840 aaaccacaag aggatttctg acacattgga ggttgttagg aatccaatttccaacaatga 9900 atgtttcttt ttacaccact ataaaagctt ggagcccttg ttaaaagagccctctcccct 9960 caagaagata tgaggcttta ttcgaaaact ttggcactgt cccatttttcctgtaagaac 10020 tttaaggatg tgagaccagg gagacaggag gttaaatgag aagggctggaaggcaaagta 10080 agaacagctg gagttcatta gctaaaatcc agggtcacta gctaaaaaggcaaccgaaag 10140 gcacgtgcag gaaaactgaa caagtaatgc agccctcttt aaaaagccttgaagcaggaa 10200 ttgcttttcc tgaacaattt ggctgccctg atggtatagc agccaaagatttattaagta 10260 tgattttact acatatatgg tctctttcta tacaggtaga atacatgtggcaatttacta 10320 gtctggtcat ttggagtact attttcattt gaccttaaca tgtgatattatgaaactagc 10380 aaaagtatga acagcactaa ggaacatttt tttttttttt ttttgagacgaagttttgct 10440 cttgttgccc aggctggagt gcaatggcac aatcttggct tactgcaacctctgccttcg 10500 gggttcaagc aattctcctg cctcagcctc cggagtagct gggattacaggcatgtgcca 10560 ccacacccag ctaattttgt atttttagta gagacagggt ttccccatgttggccaggct 10620 ggtcttgaac tcctgacctc aagtgatctg cgtgtctcag cctcccaagggaaatatatc 10680 ttaatacatg tgtcagtgct tttcatactt ctttcaatcc tcttaacaatctttagagat 10740 agatattatt aatattattc cactatatgg tggtgattca aaccaaatctctctgattca 10800 aaaattcata ggctttctac gcacccactg tagaaatatt catttagcacctactatgac 10860 caggtactct gccgaactgc tagatacaca gcaatacaca aaatagatgtgttccctacc 10920 accctcattc ctttgctaat taagaaaagc agaggccttc atagtgccttggaaatctct 10980 cataattgac tctagaattg tattttaagt gttgattttt acaactaggaggaaatactt 11040 tcatttgaat aggctaatgt gttatgtttt tacatagtac aacatttcttagttttatga 11100 aactttatag caatatctta atataatgtg cattgtttta aatatttttgttcaagtggt 11160 caacttttgg tttaaactga ggactttcag cctgttaata gcatttttcttaggaaggag 11220 tcatataact aatctttttt gaggacaagg catatgacat aatctcccccttcccctaca 11280 taatgtatat ttttaaaacc tttataccaa ccctaggaag taaaatgtgctatttttgtt 11340 gtagagataa agaaattcta gcctcagaga ggttagttaa cttgtctgaggtcacagaga 11400 tagtaatcag agttgttaga atccatttct attctattta aaatcccttctactttatta 11460 tgatgaattt ggaaatgctt aactaaagta tttattgttt agcaacagtaaaaataaaaa 11520 tagaaatctg tttttattat acattttata taaacgttaa ggaaaatgcagaagaagtat 11580 ttttttaatc tttaatttta gattcaaggg gtacatgtcc aggtttgttacatgagtata 11640 ttgcatgatg ctgaggtatc ttgtcaccca aatagtgagt atagtacctgataggtagtt 11700 tttcaacccg tgtccctctc ccttcctctc cccttttgga gtccctggtgtagtgtctat 11760 tattcccatc ttatgtctgt gtgttcccaa tacccccagt tattagctttcacttgtaag 11820 tgagaacatg tggtatttgt tttctgttcc tgggttaatt cacttaggataatggcctcc 11880 atctgcatcc atgttgctgc taaggaaatg gttttttttt ttttttttttttgtggctgc 11940 atagtgtttt atggtgccag tgtacaaatt ttctttatcc aatccaccattgctgggcac 12000 ctaggttgag tccatgtctt tgctattgtg aatagtgctg tgacgaacataaaagtctag 12060 gtgtcttttt gacagaacga tttattttcc tttgggtata tacccaggaatggaattgct 12120 gggtcaaatg gtaattctgt ttttggtttt tttgaggcag gagatgggactcgactccag 12180 agatggggct tgaacactaa accaaattta ggactagcca aaacagggcctggggggagg 12240 cagctttcca gaagacacac ccaccagtgt gccatgtcag tttaccattgccatggcaac 12300 acctgaaagt taccaccctt tcccgtagca acaacctgac aacctggaattaccactctt 12360 ttcctaaaac tttctgcata aactgcccct taatttgcat ataactaaaagtgggtataa 12420 atataactgt agagctacct atgagctgct actctgggca cactgcctatgtggcagccc 12480 tgctctgcaa ggagaggtac acccgctgct gctgaacact gctgcttcaataaaagctgc 12540 tgtctaacac cacaggctca cccttgaatt ctttcctggg tgaagccaagaaccctccca 12600 ggctaagccc cagttttggg acttgcctgc cctgcctcac tttgagaaatttctaaactg 12660 ttttccacag tggctgaact aattaacatt cccacccaca gtgtataagcactccctttt 12720 cttctcaagc ttaccagcat ccattaactt tttacttcta aataatagcctttttgactg 12780 gtgtgagatg gtatctcatt gaggttttga tttgcatttc tctgatgattcgtgatgttg 12840 agcaattttt tcatatgttt gttggccact tgtgtgtcca aaagaaatattttaaagaaa 12900 ataatacatc atgttgtata ttcatcaatt ctgattctat cattgattctacagtgccgg 12960 taattgcagt gtttaaatta gaaacagtct cagctaagaa tcttttaagatcattctcta 13020 gtagaaaaac attacaaagt aatgattccc aatccatata tgagaaaactgagccaaaaa 13080 taggctaagg agcctcccta aggtcataca atgaggcagg ggaggaggctgattagaact 13140 tctgaattgc caatgaccac aaatagtcta gggtaggcct ggttgacagaaagtctgcca 13200 ttgaacacca tcatatcaca tgacaaatac agcaaattca ttgtgcatagttacgtcttt 13260 ataaaacaaa ataatgccag gataatggta tgtgatcagc attacaattccaaagatacc 13320 aagacaacta cttatctgac acttgtctta gtatttctct aacatttatctaaaattatt 13380 tcaattattt cttttctcgg aatgcataac ttgactcatt gacttgatttatgattctca 13440 gatcaaagga aatgtaacaa cagggactag aaacactttt ttattcaatgtccaatgagg 13500 gttggggagg actccatcat tgactcatta tataattcct cataaactcattacaattgg 13560 cctggctttc attaattcat gagcacttat tgagcaccac atgccaggcctgtgctagtg 13620 ctggagatgc aaagacaagg gcaagttcaa tccatgccct caatgagtttacagcctaaa 13680 gacgactttg actaccaggc cttcattaca tagagcgaca tcctaggacttggagaatca 13740 gctttcctct ggagccttaa agacatccct atttactttt gtgtcttttctttgaagaaa 13800 aacaaaaata agtatacata ggatacatta ataataaaaa aacagtattttatgagactc 13860 agaatgctaa ttttaggatc tttgcccttc tcagttgact tttgtgtccctcaactgttt 13920 agtctgcagg acagatatca catcctgctg tgcagtttat aaaatgtccttaaaattaga 13980 agaaagaaag gccttgtctt cctgggttta agacccacac atctgaggctgtaggcattt 14040 cagatccctc tggtggatgg accaaaatga taaacaatac tgtgagataaatgctttaaa 14100 catcatctgc tctttcatct gaattcccta ttcattattc ggcaacattcacagttttca 14160 tataacgatt tcagtagttc tagggcacca gaaaagcagt actaggaatggccataaagc 14220 atagaatatt tataatctaa tgagggagac aactaaaaga aagaaggaataaaagcatct 14280 tcaacagaaa caccctttac caaccaacta gaggtataga aatgatattaggtaattagt 14340 gaccactaat ttaaagataa atatttattg agtgccagac attgttccaggcactgagta 14400 tatagcaata agcaaaaaaa acaaaacaaa acaaaacaaa agtgcccactctcaatggag 14460 tttatattct caattgtgga gacagacaat aaacaaatat ttatatataaaatgtcagat 14520 ggtggtgaca ggcactatgg aaaagaataa agcagggccc agagagagagggtaggatgg 14580 ggtagaggtg ggatggggtg gagggctgct gaggtgggat ggagtagagggctgctatct 14640 cacctagaat ggtcaaggaa gtctgcacct atatgtatca cttgagcggaggctctgaag 14700 aaagtgaggg aggatgaagg cagagaggtg agaagagagg attacaggaaaagacattgg 14760 caagtgtaaa atcctggggt ggaaatgtgt ttgcaagtgt gtctaaggaacagctaggag 14820 gccagtgagg ctaaagccaa gtgagcaaag atgggagtgt gaggagatgacaggtcacga 14880 tgggcacagc caacagtagg gtgggcagga aatcgcaagt cctttgaatttactctgcag 14940 gagatgagag gccactggag ggtttggaac caggaggcac atgccctaactcatttgaga 15000 aggatagcag tgtctggctg tcctgtgaag aagtggccat aggaggaaagcagggaagca 15060 ggcatttgca ataattcagc caacatatga tagtggcttg gtccagggtgctggcagaag 15120 atatggcaag ggaggggttc tggacaattt ggaaggtaat gccaatagatttgtatgtga 15180 taaaaagttg agaggacttg acgtgtacga gtggttaatc ttcataaaatggatgaatgg 15240 ttaaaaagat ttccgcaaag aaactgtggg ttgaaggtaa aactagtaactccaatgtaa 15300 gtgaacaaca gagaaataca aaacagacat ttttcctact cctacaaaaactgtaattat 15360 caagaagacg acatgaagtt tatacccagt attgttagca ggaagcctcattccaagtag 15420 atatttttcc ttggccattt tagcaagtga gagcatgagg ccatcataatgaacaaatca 15480 tgccatcatg atttaaaaag aagcatctgg agttttagta atatagttaggtgagactaa 15540 aattatacta aacataaaat taaaatatct taacaatatt cttagcaatttcagctttac 15600 catatccttt tgaaatctaa ttttgctata tgctttgtaa cataggggtgggggaaagag 15660 agaaatttat gagataattt ataaataaaa atacacctaa agtataagcattctcaactg 15720 atggtcagaa aatatggaag gtattcaaaa ctctagcaga aacataccataaacaagatt 15780 ttaagactga aagtagacgt ttagtggggt tcagggtgaa aggcaggggcaagaagctgg 15840 caagaagagg gaagggatac taattctaat ttgcctctgt aatgctttacatttaccaag 15900 gttccacaaa tggtatctga ttccatcctc atatcaaccc tatgaagtaagtcagaaaag 15960 acgatgtctc ttttcctaag gaatgaattg agacttaggt tgagatactctccagagctt 16020 actcagatag gaagtgacag ggccaggatt catattaggg cttctggctccacagacagt 16080 tctccttaag actttcaata aatatgtttg acaaattaag tgcttactctcggctgagtg 16140 tggtactagg tggtgtggca gcatctcaaa aagggggaaa gtcactccctcaattcccat 16200 gtggccttca gtctgagact agggagatta aacagatgcc tgagaagctgtttattacat 16260 ttacaaagca acacatttgt caaagtgaaa taataaattt agcccataaggactctgggg 16320 gcaaaaagta aaaattaagg cattagtcat tacagcaaat aaggttaacaggtgtgatgg 16380 agctccttcg gcgtaagtca gcttaaattg acaagtaaag agagaaattcactggctcac 16440 agatctgata actacaggct ggtagggcat aagcaatatc atcaggaagccgtgtctctc 16500 attacccaac actggtttgc tgtgcattca ttttattccc aggcatgttgtcaccaggtg 16560 ttggtaatct gaccccagca actcctggct aaatcccaca ggtttagctctcacaataga 16620 aaagaaagca cttcttttct aatggcacca gcaaaacagg gtctgccaaacttgggtttt 16680 gtgcctgtct ctgaaccaat cactagggta taggggagtg ccgtgctctgatggccagcc 16740 ctgggtcata tgcccattct tgggtagagg ccgggtcagt tccaccagatgagcatggtc 16800 tgaggaagaa gacggttgtt tttccagggg aaaatagaag tgcccccgctagaagggaga 16860 atggctgtca ggagggcaaa acgacagatt cactaaaata ggttgatgcctaaagaaaat 16920 aattttattc ctaaatttaa gggagtattt cagttgtttt taatcttatggaattctaca 16980 ctgggaggga gttggtgcag gagattcatg atatgcaggc ataggctacagaataatgct 17040 ttgagttttt atcctttact tttcctttcc tttaagcttt aaagacacgatttcttcatg 17100 cagggttgcc ctgaggtgag cctcatcatc tctttttttt gagatggagtctcgctctgt 17160 cacccaggcc agagtgcagt ggtgcaatct tggctcactg caacctccacctcccaggtt 17220 caagtgattc tcttgcctca gcttcccgag tggctgggat tacaggtgtgcaccaccagg 17280 ccccaccacg cccggctaat ttttgtattt ttagtagaga cggggtttcaccgcgttggc 17340 caggctggtc tcaaactcct gacctcaggt gatccaccca cctcggcctccctgagtgct 17400 gggatcacaa gcatgcgcta ccacgcccgg cctcatggtc tctttattgtaccttttcta 17460 gtctctgctt tcctgaagcc agaggtcttc ctatctccag aagctccaaagacacacttt 17520 caaacccctc ccagtcactt ggccttttct gatgacttct ttccttcaaggctgccttta 17580 gtaaccgatt attgaagagg caagagaaag ccctcagcct tctccactttcacctccctg 17640 ggctccccaa gtttggccga ctcctctttt caagttcaca ttttctcctttccacagagg 17700 tttgcaacat tacctttaag aaatcatctc cagtctctat cacgtttcaacagttcttta 17760 ccccatgctt ttatccctgt ctcccaccaa tcatatccac cggccctattgaccgcttgt 17820 gggagttaga attttggaga ctggtcatat gtcacaaagt cctgctctagaaggcagaac 17880 actccatttc ctgctcctcc aaagcccttt atctctccag gcctctcctcctgtagctct 17940 gaagctggat tgatgagatt cccagagggg agcatttagt gctctgagtgctttgatgaa 18000 attgattagg taaatggaaa catatttttt gcaaccactc tagcctgtagaaacaataag 18060 ttgcaatgat ttgccatttt tgaaataatg aaggttcttt gtaattttaaatattctttt 18120 gccacaagag attgttttcc agcagtaaaa taaccagaat gtttgatttgaaatgttgaa 18180 aaaatatata ccgtctgata tctttagagc agcactttca ttatcaatgatggatttaac 18240 attttgttta atttttctag cttctctcct gaagggtttg aaacatgccaatattgtgct 18300 cctgcatgac ataatccaca ccaaagagac actgacattc gtttttgaatacatggtgag 18360 ttgttcgagc attttacaac acttgagaaa aataacctgg tacttgtataatgaatctgt 18420 taatatttta tggcatgata aaacttttat tataatgtga aaagtatcatggaaattttc 18480 attattgtga ttagtagaac cttattgttc ccacatccat ctttggtcctgcttccttac 18540 ccatgacttt tgctgtccct tttcccctca tcagcaataa taaatgaggatcttgagttt 18600 accttctaaa taaaactttt gcacttattt ttaatctaat tttaatcactatctgagcag 18660 aatccaacat tttttcattg acaataaagg taaaaatcac aagatatttaaaaattgtat 18720 gcaagcttgc taaagaataa ctcatgttgt atttttggaa gaaaaaatatttaaataagc 18780 agaaagaact tataaggtat gtgtacttga cttgcctcca aggacacttggagagtgaaa 18840 aattcctgcg tcgttgtgtt cagtgccagt catttaaaat gagcatctctgtgctgagaa 18900 acaggctttg ttctaagagc agccagttag aaagacacac tgtgtttgaccttaacagtg 18960 ggttctcaga aaacctggtt atattccttt tgcaccttat tcttaaaattctgtacttcg 19020 tgataccttc tgacagtcaa gtcaatgttc tgctttagga tgctatctaagcaccactaa 19080 attcactcac ttctctttct ccgctgtttt atttagcaca cagacctggcccagtatatg 19140 tctcagcatc caggagggct tcatcctcat aatgtcagag tgagtacgttaagggtcagg 19200 accctctcct ggcttgccca cagaaggaga attctgaaac agactgtctcacaaagcaaa 19260 gtcctatgat actaaataag aggatggaca tcactgatat tccagaaaaaagttttgttt 19320 tgttttcgtt tttgtttttt tttaaaaagg aaagaaaaaa gaaaaagagttgctgagttg 19380 cttcttaaga tatggagcaa tgttttctga gcaacctaat gctgtcagtcatggctacat 19440 gcaaatgtgc ctttagatga ataaacgagt gaaggagaat tatactaaaaggaaaaaagt 19500 aaagctaggc catcaaaaaa taaatacctt cttcatatca gattactgtggtctaaggtg 19560 aagtctgcaa tacttgtact agcagatcct attatatatg tggccctaactcccattttt 19620 ccagtcatta gaatcaaaat aataaactct taattagcta taattctacatctgttataa 19680 attttagaaa ccatttatat ttcatacttt tcattcccta aggttttattggcattaatt 19740 aattgattgg ctcttaaaat aaccgtatga aatttgtata tgatgtatttattcatttaa 19800 ctaatattta tttatgtatt catttattca ttcatttaag aaatatttattgagtactta 19860 ttgcgtaata agttctgggg tttcaataat gaataagttc tgtttcttattttcaatgag 19920 cttaaagtcc agtaagatat atgaacttaa ataggcagtg agggccagtcttcaagcaac 19980 agcaatgcaa gatggcagcc accatgggct caggcaattt atgaaagccaaatatacagc 20040 cttaaaatag aatgtggacc taaataccca gaagaactcc cctttgtaagatttgtaaca 20100 aaaattaata tgagtagagt taatagttct aatggaatgg tgaacccaagagccatatca 20160 gcgctagcaa aatggcagaa ttcatatatc atcaaagtta tccttcaagagcttcagcgc 20220 ctaatgatgt ctaaagaaaa tgtgaaacgc cctcagccat ctgaaggacagtgttacagc 20280 aattgatcaa aaagaaaaac cacaggccct tccccttccc ccatacttgatgtaagcagt 20340 cttcattttc catagtagta aattttctag atacagcttg tagagctcaaagtactggaa 20400 agaaagctcc cattcaaagg aaatttatct taagatactg taaatgatactaatttttgt 20460 acatttggaa tatataagtt gttagcctgg cgcggtggct cacgcctgtaatcccagccc 20520 tttgggaggc cagagtgggc agatcatgag gtcaggagtt tgagaccagcctagccaaca 20580 tggtgaaacc ccgtctctac taaagataca aaaaattagc caggtgtggtggcgcacacc 20640 tgtaacccca gctgctcgag agagtgaggc aggagaattg cttgaacccaggaggcagag 20700 gtgcagcgag caaagatcac accaatgcac tgtagcctgg atgacagggcaagactccaa 20760 ctcaaaaaaa aaaaaaaaaa agaaatatgt aagttgtgct ataacaaataaataggcagt 20820 gagaagcaaa gtgctaaagc ctatgaccat ggtaactagg aatactgtgggaacacataa 20880 taagggaacc taacccagtc ctggaagtaa ggttttggaa aggaatgtttgaggacaaag 20940 ggttaaagag agtgaaaaaa aaaattaaaa taccagttta gctgtgtggagaatgggata 21000 gggagctaac tagagaaatc aaataggaat gtttcatggt atgttaaggaccctggtaag 21060 ggtgaagacc attacattat ctgcaccatc gcgggacttt ttttttatggtaatgcttgg 21120 caatttaaat agaggagcag agaatgtaga cagttggatt gagtcagagttgaagttctg 21180 ccagacatgt gaaaggaaga gacaggtagg caagagagtt gaagagattatcaagacaga 21240 agttaatgtg ctggccagtg gcatctagtc tgagtctaat ctgagggaaggaagtgaaga 21300 taagcagctt gctgatagtt atgaagagag tggaaggctt caaggacctacaggtgttga 21360 ttaaatagaa gaatgattgg agaaagaata actgtgagag agtgagattttcaggcttga 21420 gtgactctca cataccagac actgtgctaa atgcttcaaa gacatgatccctgccctcaa 21480 gggacttaca gccaaaaaca agagataaga aatacacacc aatactattataggacactt 21540 gtgtagaata tcaagaaaga aatacgatct agtactgtag atgtgcaacggcatcaaaga 21600 tatcttctag tttcaagaag tttcagatcg gccgggcgcg gtggctcacgcctgtaatcc 21660 cagcactttg ggaggccgag gcgggtggat cacaaggtca ggagatcaagaccatcctgg 21720 ttaacacggt gaaaccccgt ctctacaaaa aatataaaaa attagccaggcgtggtggcg 21780 ggcgcctgta gtcccagcta ctcaggaggc tgaggcagga gaatggcgtgaacccgggag 21840 gtagagtttg cgtgagccga gatcgcgcca ctgcgctcca gcctgggcgacagagtgaga 21900 ctgcgtctca aaaaaaaaaa aaaaaaaaaa aaagtttcag atcttaaacacactgcattt 21960 caacagtcta gaataggaga gcatgttaca gggagagaaa atgttttcagcaaaggtaca 22020 gagtagggaa atagaggata tgttcaagga agaggacccc agagtcatggtttgttaggg 22080 ttagaggaaa cacagtgttt tgcaatctcc aggttccatt agtgcgttatgaaatcaata 22140 tggtggttag caacctgcat tttaaaaaat gaaataaatg gatgagaagagaatagaaaa 22200 tattagcatg cattacattt tgaaagagca agtattattt tctgcaacttttgctccaat 22260 tgtaactgta cttatatttt tatgtatgga tgtgaatacc agatacatatatatttctta 22320 ctgtagactg cagtcaaaaa atctttaaag cactggcctg gtctaacttccttattttgc 22380 agaggagaaa tccaagatct gagaggacaa acattttgcc tgaggttatagaaccagctt 22440 atgccattgc taaaagtgat tcttagttaa aattctttcc cactagtgccatactgcact 22500 tctagttctg ttggcctgaa atacagaata tattagtgaa acagcatacacaagtctggg 22560 gaaatatatt gggtaggtgg ctgagagcct cattttctaa gaaatgtggaccttaggcag 22620 ggtatggtgg ctcacaccta taattccagc actttgggag gccaagtcaagaagatcgct 22680 tgaacccaag agttcaagac tagcatgggc aacatagcaa gacctcatctctacaaaaaa 22740 tttaaaaatc agctgagcat ggtggcatac gcctgtagtc ccacctacctgggaagctag 22800 gtgggtggat cgcttgacac aggagtttga ggctaaggtg agccatgatcacacaactgc 22860 actccagctt gagtgacaga ggaagaccct gtccctaaaa aagaaagaaatgtggatttt 22920 attccttaga cagtacagtc attagtcatt aagtttgagt tgagagaaaataatatgatc 22980 agaagaaatt tatatcactg tggtctgtag gatatatgaa aggaaataagagactagagt 23040 cagggattcc acttaagtgt ttgtttgttt gttttgagac agagtctctttttgttaccc 23100 aggctagagt gcaatggtgc agtcatggct caccgcagcc tcaaactcccagcctcaaat 23160 tatcttccca gctcggcctc ccaaagtgct ggaattacag gtgtgagccaaagggtttat 23220 tgatgtggtc tggcctagtg cctctcaaac ttcagtgagc agacaagtgaccgggaacct 23280 gactcaacaa gtctgggttt aagcctgagc ctctgcattc taacatgagtcaagctgatg 23340 cagatggtgc tggtcaagag ccaagcactg agcagcaagg atctagttagcaattagtaa 23400 tcaaggttga tattatggta gtgacaataa gaatggagag gaatgtgaaaatcagtaaca 23460 aagaagagtt cacctcttgg taatgtgagc atgaggaggg aaaggatggggccaaacata 23520 actggttttg tgtttgactg acgaggagaa ttgtagctct attaacagaaataggagaag 23580 aagttggttt ggagagaaag aggagtcctg tttcagacgt gttgaggtcccaggtgagac 23640 aggatctcca aagggaaatg agcagtaggc aacctaaaag gaaatctgtgctcagaaggg 23700 agctgtgagc tcgacgtgta gatctgaggg tcatcagcac atagagtttagaagacaagg 23760 agtaggcaac caaaagagca aatacacaaa gagaggagga ctgatgatgagacttttgcc 23820 ttttaggatg agaagaggaa caggaaatga aggaatgaag ggaagcagcttgtaggaatg 23880 tagagcatct gaaaaaaaaa tacacactgt catggaagtc aagggaagaagaatttcaag 23940 aaggagggta tggtggacag tattacaagc atcaggaata cagctaaaagtcatactctt 24000 gactgcattg accttgtgga tttgtgaggg acacactaat aaataaaggaatttattgtg 24060 ggtatatgga ggcacaaagg aagaggttat ccaaatcaaa gcaggtgggagtagggatga 24120 gttctccaag gtggaggcat cagtgaatgt gggaaggggc acagagcatccatgcccatc 24180 ccaggcaagc caccctccag aagcctccat gagagttcag ctatccagaaggtctctgta 24240 ccctaatctt tctgggtttt gcataggctt cattgtgtag gcatgatttattaaactatt 24300 ggccactggt gatcaactta accttcaacc cctctcccct ccctaatcatgccttggtct 24360 ttccagtgac cagtccctat cctaagctac ccaatggtct gccagctatcagtcaactct 24420 acaaaaagac atcactttgg agattctaag gattttagga gttggctgtcaggaatttag 24480 ttgaagatca aatatatatt tcacaatatc acagtcgtgc tattttatatcaggcgccat 24540 taaatggttt taaacaaaga ggtgataaat tcagattttc tttttataaagcttacactg 24600 atgacagtgt ggtgaataga ttgggatgag ggcaatactt tttttttgaaatgttatatt 24660 cccctgaccc tactttctcc ttgttttctt ctacctctct ccccctactcacacagaaaa 24720 cttctctccc tctactcatt ccctgaatgc tggtgtctgt taaggttccagccttgacag 24780 tgaggctaat cagaaccaca gtggtacaga tgtgagatga tggtgggagaaagtggacag 24840 atatgagacc aattacttag ccggaactga cgggaaaaac aagagtcagcgatatttttt 24900 tctggatctg agtattaaaa tggatgatgg tgccattcac tgtgatagagaatcagaaag 24960 aaaaatttat tttggagaga taccatgaat tgtgttttag acatgctaagtttgaggtga 25020 ttatgggatg tacaggcgag ctccagactg tgtgggccta aagtagaaaggcaatctgag 25080 ttggagataa agattttgaa atcatcagaa tacggttgtt cattagagcactgtcagtgg 25140 gtaagatagc taagggagca tgtgtagagt gataacagaa gatcaaagacggaaccctaa 25200 gaataacaat atgttattat ttattatttt attatgtttt attttttaattttattttta 25260 tttatttatt tatttttaga cgggagtctc gctctgctgc ccaggctggagtgcagtggc 25320 gcaaactcag ctcactgcaa cctccgcttc ctgggttcaa gggagcctcctgcctcagcc 25380 tctcaagtag ctgggactac aggcacccac cacctcacct gactaatttttgtattttta 25440 gtagagacgg ggtttcacca tgttggccag gctggtcttg aacttctgaccttgagtgat 25500 tcacctgcct tggccttcca aagtgctggg attacaggta tgagccactgtgcctggcct 25560 atttttgttt tttatagaga tggggtcttg ctatgttgcc caggctggtctcgaactcct 25620 ggactcaagc aatcctcctg ccttggcctc tcaaagttct gggattacacatgtgagtcc 25680 ctgcgcctgg ccagaatatc aatatattag attttagtag aagtagaacctatgaaaaga 25740 acagccagag gggcagaaga aaaattagga gattgtggaa ccaaaagaagagagtgcctc 25800 aggaaggaag gcatggtcta tgatgccaaa tgctgcaaag ataaggaataagaagtatcc 25860 attgggtttc ataggaaaag tcatgggaaa ccatggtaaa aaaacattgtgaatgacaca 25920 atcgttgcaa aagcattttt atagggggat gaattttgta tttcagaggacaaacagttc 25980 catacaatgg caagatctag tgtgtgacca cgggagttag tgtctgaagtggattggaga 26040 agcagatcat tggagctgag gttggctaga gctgttctca tggacactaatgtcatggag 26100 tcaacagctg tgatccaagt gcccacatct tcagtgaatg acagagagggattgagagtt 26160 cagtgaatga ccgctaaaag aagagtaatg gaagatgtgg ctggatggcattaaaatcca 26220 agggacaggg gtttttactt aaaagtagag aagtaatggt tttgaagtggtagtggggaa 26280 aagggaggca gcttatgaca cttgtcagtg gtcaaaggta tgaggaagttatagaaaaac 26340 taacatccac ttgagaatat tatagggaag cagtgagctc aaggtctcatttaaggaaag 26400 gagccaaaag gaaattcacc agaggttagc ttttaggtag tttttaaagcaggattgaag 26460 aatggagact aaacagtgaa aatgtttggg agagagagga gcaatagatatgaggctaaa 26520 cagaggaagc acagaacaga atggagatga gtatgttggg aggaaaaggaatagtcagag 26580 gcttatattt tgagttgtga ccaaggaaga cagggtggga atcctcgtgaggttatcttg 26640 tttcagattt ctagtagaat gagtcccagg gattccaggg gggatggaaggactcaggct 26700 tccctataag gagttggcta acggatctca ttggtttttg agtaactcctggcccagatg 26760 gcactagttc aatggaatta ttttgttccc ccaaaactta ttgagttggaaacaggtcta 26820 actcctggga tctgggaagc ctttctggaa agagtcaccc acgatctggctgatgttgaa 26880 ctgtgcagac accatcatat ttggttatgt taggatgcaa taattggtgaagcttctgta 26940 gtgttgaatg aagaatccag gttggaaggg atgaaagggt gagtgggtgatgaggtttgt 27000 cagcacagac tgcaattttg agaaatgtgg ttataaaata ccataccttaataccgcagt 27060 gctttaccac tcacaaatgc ctgtagacgt atctggcaga gaggaaaggggttgaatggc 27120 aagaatgtgg gaagggactg tggctagtta gtgaaaatag tctacacttgggacataaaa 27180 ggcatttcaa gctgacctac taagaagctc tgtctctgac tcagccagctggctctctcc 27240 ttccctgtca tgttttcatt ttctgtcttt tctctagttt ctcaggatggtatagtggag 27300 tcagacaagt ctgaatttga gtcttggctc tgactattcc tagacatgttttaaaagtta 27360 cattgagccc tggttttctc tgtaaactga ggataagcat gctatcccaaaggttgtatc 27420 cctcactggt caccagcttc ctgtcttcta tccacctgtc ttcctcttcctctttcccta 27480 gtcctgcata ttgaaaaaca tttttttttt tttttgagat ggagtcttgctctgccaccc 27540 aggctggagt gcagaggcac gatcctggct cactgcaacc tctgccttccaggttcaagc 27600 aattctcctg cctcagcctc ccgagtagct gggattataa gcatataccaccacatctgg 27660 ctaatttttg tatttttagt agagatggag tttcaccaca ttggccaggctggtctcgaa 27720 ctcctgacct caggtgatcg gctcgctttg gccttccaaa gtgctgggattataggcgtg 27780 ggccactgcg ccagtctgaa aaacgtattt ttaagcacat actatcgtatcttcttgtct 27840 tttacctgga atttaagctg gttgtttgta ttaccttttc catggacatttatatttata 27900 accaatcaga aggtttaaat gtcagtgtag gaattttgtg ctatggaagcttcgtggctt 27960 ggtgaatggt aaaatgaata atgtgtgtat atttgaagca tcagaaagagaaaatgctgg 28020 gaagattcat agaaccagtt aacatttgaa ctaggagtca taagaaatttttaaaattct 28080 taaatggttt atgaacctga tgtggtagct acatgaaacc tgcatagctgcaggtatgct 28140 atggtaggta aactctccat gctcctgctt ccattggacc atttggctccaatgtctcca 28200 ggtctttgtt agatcaatac tggtcctagc atctctgaaa gtcctagctttctaagatgc 28260 tgttgaaaaa gaggattaat ccacataact ctgcatctgc cattttgcccatgtcccagg 28320 aatgctgggc ctagcccttc ctttctgaac tgccagaaca cgttctcagttgacatacgt 28380 ctttgtaaat actgatgttg gtgtttgaat tctcaattgc caatggcactggaaaatagc 28440 aaaagatact tggaatacta agcattcttt ttttcccgta agtttctgtagtgatgggaa 28500 cctagtaatg gctttggttt ctgtgcctca taaccacatg aaacatttttaatttggggc 28560 tcagaatgtg tttttccctt ttatttctcc accactacca tttaccctttctcccttctt 28620 cctcctacaa tttgttcctt attctttttt gatttttttt gaggggggggggtctaactt 28680 attttggtct ctcttccctt ttcatctgta ctgtgtattt cccttgttttcaactttgaa 28740 tttaagactt taaaaatagc tttaaaaaga taaagatttc tttattttctaataccatct 28800 aaagatatat tttttagtgt ggtctccttg tgttgtgttt ttaaaagggtttcatattgg 28860 agagcctgga aaacttaagc agttgtaaac tttagaatat catttccaggtcaactttga 28920 tcttatatgc caagttcatc ggtggggaaa aaaattaaat ctttcacatctaaatcaata 28980 actagtgttc caaaggaaac ttcaaagttt cactttagat ttttaaagaagggtaattcc 29040 ttcagtatca aagaaatgag atgtcaggaa aagccagaat ccctttgtttaggacacagt 29100 ctagttactt gacttttctt gtcctttttc ttccccctct gaatgtaaaaatcttcttct 29160 tcttcttttt tttttttttt ttggtctctc aagagacact tttactatattctttgagat 29220 gactgttttt gatttagagg cgaaatcagc acgtggtggc tcaaatctccttatggatag 29280 tgtttcttcc ttccagcttt tcatgtttca acttttgcgg ggcctggcgtacatccacca 29340 ccaacacgtt cttcacaggg acctgaaacc tcagaactta ctcatcagtcacctgggaga 29400 gctcaaactg gctgattttg gtaagtcgcc cctcgggtct cattctgggctgtgaacaat 29460 gatgcttttg tgtgcacttg tttaagcgtt gactgggcct ggcctttgaaaactggaggc 29520 ccaagaacat gatgctttgt gaggatatca aactaccaca aaggaagtgtgaggcacgaa 29580 acagggaggg attggtagct ttctaggatt ccaccaagtc ccagtttagtcagatggcca 29640 aaagctgggc acccttgctg ccccactgcc agttttgata tagagacattggtagagtaa 29700 actgtactta gtaagttttc ctaaatctaa gtgaatatac aaattatattggaatagatt 29760 gagattatcc caagatgata aagaggttaa ccccagattg tagcatggactcctgtcagg 29820 atggagactc caggacactt gttcctgctc tcctaccttc tttatataagtgtgagatgc 29880 aaagttttat tcccattaaa gtgaagcaga tttcctctaa gtatcactgtatccttccat 29940 tttagcactt atcgcagttt ataattatat tcacacacat aaatacatacatgcatacat 30000 acaaatatat atacatgtgt gagcacaccc ccacacacaa atatatatagatttgcgtga 30060 tgattttgtc tcaactggac tgtaagcata atgagggcag cctgggtttgtttttgctta 30120 tcattttatc cttagtgcct ggtaccatag taggtgctta ataagtacttgttgaaaaac 30180 tggctctatg tgagctaagg aaccactctt ctctgtttgg cagatgccaaatggtgatac 30240 tatcactgca gtatttattc tgagatggca gcttttatcc tgacatgtaagcatttaaca 30300 gatatttgtt tatcaattct ccacaatagc aaactcatct attgaagtttttcccaacaa 30360 tagatcatgc aattctgtga gataaacagc tgactgacag aaagactcattttgcagaac 30420 agtacttaga aattcatcta aggtcctacc aaactaatta atttggatgagcagtcccta 30480 ccgtttatct actaaactgg gctttcctgg agtgccaaaa cggaaggtggccatgttagt 30540 catgaacagc tcagtttctg ttacagagac ccaaaattac agaggtataacatgctagaa 30600 acttaacttt ctttcgcatc acagtcctga cctaagcagg cagagcatgtatggtggccc 30660 catgctatct tggcccaggc tgcttctgtc acgtggctcc tccatccccaattgtatgtt 30720 tcaagatggc tgccacttcc tgctcatcac agcccagagg agggagaaaagagaagcaga 30780 acccttaacc cctccactaa ggcataatct ggaagttcac acatcacctctgttcatatc 30840 atataggcaa gaacttagtc acctgaccac acccagctgc caagaaggccacatctagct 30900 gcaaagcagg ccaaaatttg agaaattcac ttgatgaagt gatagacaagagtcaagata 30960 gtgattagtt ctactaaaag cacctaaagt ttgtgtgtta ttttttctaatggtgtttac 31020 cctggtccag tgcatcatgg tgcaagccaa ggtccagaac gatgggttttatgcttttcc 31080 cttttggaca ggtcttgccc gggccaagtc cattcccagc cagacatactcttcagaagt 31140 cgtgaccctc tggtaccggc cccctgatgc tttgctggga gccactgaatattcctctga 31200 gctggacata tggtaagagt ggtgccgaga aaatgtgagt catcctactcacgagggttg 31260 ctttatcatc tacattatat tttaataata attctaaaaa tggcaatcacgtatatattt 31320 ttatatatat ttatatttat atattttata tatatttata tagttatatatttatatttt 31380 atatatttat atatttatat atatttgtat atatttatat atttatatatttttatatat 31440 ttattatatt tatattttta tatttttata tatttatata tattttatatatatttatat 31500 atatattata tatatttata tttatatata tttatatatt tatatatatttatatattta 31560 tatatattat atattttata tatttatata ttatatatat tttatatatttatatattta 31620 tatattatat atattttttt atatatatat atatgtattt tttttttttgagatggagtc 31680 tcactctatt gcccaggctg gagtgcagtg gcacgatctc agctcactgcaacctccacc 31740 tcccagattc aagcaattct cctgcctcag ccttctgagt agctctactaaaaaaatact 31800 aatatttgta gaagattctt gcaattattc tataaccttt tactgttgaactgagaccca 31860 cagagttcct gcccaaggca tcttctgaat ctgacactct ttttatgttattttattttt 31920 tgagattggg gtcttgctat attgtccagg ctggtcttga gctcccaggctgaagcagtt 31980 ctcccacttc agcctcttga gtagctggga ctatagggct gcaccactgcaccctggcaa 32040 tctcatgctc tttctttcac gcctttcctc ctagctcctc tctttaatcctttgccttgt 32100 cttctccttg acaccttatc cacagagaaa caaacatata tccccaaaccacagacacac 32160 agatgtgtgt gcacgtgcat gtgcatgcac acacatctgc atgaacatactcacacatgt 32220 ccaaacgtag ttcagagcct ggtttaggaa aaaaaaaaaa aagcataaagaccaagcttc 32280 aagacacctg attttcatgc cagttcgatt tctaatcaat taactctggattctgttatc 32340 ttgaaaaagt catgtatcct ctctgtgtct atgtttctcc atttttaaaaatgaaggtaa 32400 taaactctct ccatctgagt taaatggaat tgtagtacaa atataagaaccaaataggtg 32460 gctgggcttg ccgtctcatg cctgtaatca cagcgctttg ggagaccaaggctggaggat 32520 cgattgcttc agcccagttg tttaagatca gcctgggtag cacagtgagatgctgtctct 32580 acatttttta aaaaaattag tcaggcgtga tggctaatta aacacttcaggaggctgaag 32640 taggaggatc tcctgagcct gagaaattga ggctgcagtg agttttgatggtacccctgc 32700 aatccagcct gggttacaga gcgagacccc gtctgaaaga aagaaagaaacagagagaga 32760 gagagagaga gagagagaaa gaaaggaaaa gagaaggaga ggggagagggggagaaaggg 32820 agagggggag agagggggag aaggggagag gggggagagg tggggagggagggagggagg 32880 gaggaaggga aggaaggaag gaaaggaagg aaggaaggaa ggaaggaaggaaggaaggaa 32940 ggaaggaagg aaggaaggaa ggaaggaaag aaggaaagaa tccagataggtgctatcaag 33000 taaagccaca gagttgggga ggctctaagg ttaatgggtt acaatagtgagcatgggctg 33060 tcagacatgc atcatcctag aacggcagtg ttattttctc tggatcatgttcctggagac 33120 ttcccagtca tttgggggcc actgttagat atgtgatgac tttacagacgtagacaactc 33180 cccaaaggta aggaaatata tgaatctctt tcagtacctt ggaagaaagggtttatataa 33240 aaacacaaag ccccattttc aaaaatccat aattgatttt aaaaaattaaatggtgtcct 33300 aaaaggctaa actaagcttt tagatctccc aaagaattaa gaaaggttgcagacattttt 33360 ctccagtgta gagtcattga tttctgatac ccagtacaat ttatagaaatatcatctgct 33420 agtcaaaacc ctcctgaaac tgtcagctca caccgctcag cactgtcacttcaaaggact 33480 ccggcaggct ctggcttact cagctcttaa tgatgtcttc ctgattatgtttcacagagt 33540 gaaacttcta cccgtcaatt ttaaactaat tttattatgg aatagttaaaacattcaaga 33600 gtatatataa catatatgta gatcagtgat tctcaaccag ggagcaattttgctctgcag 33660 gggacatttg gcaatgtctg gaaacatttt ttgttttcac agctgggggtggggtggtgg 33720 ggggtatcac tggcatctag tgggtagaga ccagggatac tgctaaacatcctacagtgc 33780 agaggacagc ccctgcaaca aagatttttc caacccaaaa catctgtagtatcaagatta 33840 agaaagccga tgtaggttaa gaagcttaat ttacttttag agacagggtctcccttggtt 33900 gcccaggctg gagtacagag gtgagattgt ctcactgcag cctccaactcctgggtttaa 33960 gtgatcctcc tgcctcagcc tcctgagtag ctgggaatac aggtgtgtgccaccacacct 34020 ggctaattaa aaaaaaaaaa gtgtagagac agagtctcac tttgttgcccatgctggtct 34080 caaactcctg gcttcaagag atcctcctgc cttggccttc ccaactgctgggattacagg 34140 tataagccac cgtgcccaac caattaagaa gcttaataac gtgaacttcataacctgcta 34200 cccagtgtaa caactagaac ataatccgta ctgtcctatc aactgtgtccctttcccatc 34260 aacctgcccc tccactagaa ggccttctac caaaattttt tttccttttttcatcagtat 34320 tctcatatct ttttaaaaat aatcctttta cattttagag gtattcttaaaaatattttt 34380 ttgttttact tgattttaag ggttgttttt ttttgagacg gagtctcgctcgtcgcccag 34440 gctggagtgc agtggtgcga tctcagctca ctgcaagctc cgcctcccaggttcacgcca 34500 ttctcctgcc tcagccatga tgttatattg cttctagtct tctgtgacttggctttgttt 34560 cattcaatat gttacatgtt tctaagattc atccatgttg atctgtttagctatacttta 34620 ttttctgtta gtgaatattt catttttttt aatgtctata gctttgcaataatacttgat 34680 accttgtagg ccaagtctcc cagcctattc atcttcttca tgaggatacatcagataaac 34740 ctagtttaag ggacattcta cagagtaact gacctgtact tattggaagtgtcaagattt 34800 taaaagataa agactgagga actgttccag attaaaggag actccagaaacctgccaact 34860 aaatgtaacg catggtccta gattggatct tgggggagat ggtgctctaaagaatactgt 34920 agggactata ggtgaaattt cagtagggac tgtggattag ataggggtattggatgaatg 34980 ttaaatttcc tgattttgat aattgcactg ttgttatgta agaggatactttggttctca 35040 gaaaatacca acataattat ttagggatga agagtcatga tatctacaatttactcccta 35100 atgtttcaga aaagatatag acagacagac agacagacag acagacagatagatagataa 35160 aataacgaaa caaaagtgac aaaatattgg cgatggatga acctgtttggaggatataag 35220 agagttcttt atactgctgc aacttttcta taagtttgaa attatttcaagattaaaagt 35280 tgcctccaaa ttgcgaaatc cttgctgttt catcaaagtt agtgtaagacagcactagcc 35340 taatatgtga tcagtgtttg taatttcttc atgtgtgttt gagaagaatgtgtgtgtcca 35400 cccaaatgtt gagtgctgct ggggtttttt ttttgttttt gtttttgtttttgttttttt 35460 tgagacagag tctcactctg tctccatgcc tggaatgcag tgactcaacctcggctcact 35520 gcaacctcca cctcctgggt tcaagcgatt ctcctgcctt aacctcccaagtagctggga 35580 ttacaggagc acaccatcac acccggctaa tttttgtagt tttagtagagacggagtttc 35640 gccatgttgg ccaggctggt ttcgaacttt agatgtcagg tgatcagcctcccaaagtgt 35700 tgggattaca ggcatgagcc accgcgcctg gccaagtacc catttttacatatgttcaaa 35760 aattcaaggt tgctaattat attatccaaa tcttctttat attatttttgtctttttaac 35820 ctaccaatga aaggtgtgtt gaactcattc actatattgt tgatttgtcagaattctatc 35880 cacttttgct ttatatgctt tgaagctatt ttcactaagg gcaaataaatttaagactgc 35940 tcattattcc tttgtacact ttagttacca ctttcagaat aattttcatttctcctgaaa 36000 tacatctttt agagtgtttt gttttgtttg tgtgtgtgta ggcctgctggtggcaaattc 36060 ttcgtttttg ttttcagaag ataaacccta attattgaaa ggtggttttgttggggatgt 36120 gattctagac tgacagttat tttctctcag aactttgaag atgtcattccccttctttgt 36180 cttccattgt tgctgtcgag gagtttgctt ttagccttat tatcttccttttgcaggtga 36240 tctcattttc tctggatgtt ttaaagactt ttttctttgc ctttatgattatgcagtttt 36300 ctctaggagt tgtccagtgt ggatttcttt ttacttaccc tgtttggtatatcttgtgtt 36360 tcttccattt gtgaattcat gtctttcatc agccattttc tttttgaatattgactctat 36420 tctattctct ctctgtagag ctccaatgaa agactattag accacattcttctgttatcc 36480 atttctcttc tctccttcat attttccatt tccttaactt tctgtgatgcattctgggta 36540 atttcttcag ctcatctacc agttctttaa gtctctctta aactatgtattaggttggtg 36600 caaaagtaat tgcagttttt gccattaaaa gtaatggcaa aaccatagttgcttttgcat 36660 caacctatat ctcttacctt tttaccacat atacaaaaat gtatgttattctatgaataa 36720 gtgtttcatg aatttaacca tgagcaacaa tgacacaata taaaaatgcagttataagtc 36780 aaaattattg ttattactct tattcattcc atttgattgt tgttttcctggtaaaactaa 36840 aaatgtaatg tagaaataga acaatatgca tcttccattg agctcactatatttgtttac 36900 cctcaaagta attgctagac cttgggtatt tacactgaga tccctctcctcccatttttt 36960 tctttttctt ttcagagtga taagagggga agtgagaagg gagaagatttccagttgaca 37020 aagaatgaaa aagaaagaat aatcctattc tgctaggcca tgcaaccccatagggtccaa 37080 agtgaatgcc cttgtaggag gtagatgaca ctgggtgagc attagtgcatttgtcttaaa 37140 gaaaccaatt ataacccgta gtgcagagcc tctccttcac aatgaggcctggtggcagca 37200 gtgtcagtag ggggccagag caaataaaca ggggctctag ttaattatggaaaacttgca 37260 actaggacat attggttatt cccaaagctc ccaaccaaca ttctctcatcttctgacgtc 37320 ttttcttctc tctctttctg ctaccttttc agaccttaaa agattccattagtgacttta 37380 gtgagaaaaa tgcaatattt taggattatt aaatggtgtg gtttttagttttttgtattg 37440 tgttaaaata tacataaaat ttaccattca tcacgatttt caggtgtacaattcagtggc 37500 attcagtaca ttcacattgt tgtgtaaccg tcaccactgt ccatctccagaacttttcat 37560 catcccaaac tcaaactctg cacgtattaa atgataattt cccattaccccctctcctca 37620 gtccctggta accacgattc tgctttttat cttgatgaat ttgactattcttggtacctc 37680 atataaaagt ggaatcctac aatacctctt ctgtgtctag cttgttttgcttggcataac 37740 attttcaagg ttcatccatg tcgtagtaca ctgagttttc cagaagcatttatttcagta 37800 cacaaggtca tctattcagt atcagtttca ggcagctgct ggtgttaggactagagaaag 37860 ttgtctctgc ctaacagatc atttactgtc acatttctcg ctgcaaacttccaaatataa 37920 aaagggtggt ctagagaaaa gcaagtgaga atgtcatgtc actgccatatattacgttat 37980 tctgaattaa cttcaacagt aagaaatgaa atactgattc atttctcccaacaacatttt 38040 gatattctcc ttgcacctcc aaaaagccta aaactcccga gatggattttttttctccag 38100 ggactgccta aggaatctga ggaatctttc cccctcttat ggaagaatttgttcatgctc 38160 agaatagaga aaaagtagga ggagaaccag aaagaggaga aaacatctaagcagtttcct 38220 ctaacttgac tgaagaacca catttggaac aataaaatga cccagcacatctctcccttc 38280 tggaagggtt taatgtttga tgtcacaggg tcttttctcc cctgcatatgaatttcccct 38340 tcgtctacac gggctgcccc acgggtatct ccacacagca gaaatcctcagagaagctta 38400 aagatatgta gggtaagagg agccccagga atgaagattt aaggacaaaacagaaaaata 38460 aaaggaaata gaagctggtt ccctatctgg acttgaatgt tcagaatatttaaaatgttt 38520 gctttaagaa tagtctgtgg tgggcaaaat agatgatagc cacatgacttgtattcctaa 38580 gggtaagaag caaattaaaa aaaagaaaca gttctgaaca gaaatgaaaaaataagataa 38640 attgcatagt tctttttttt tattagatgg agtctggctc tgtcgcccaggctggagtgc 38700 agtggtgcga tgtcggctca ctgcaacctc caactccccg gttcaagtgattctcctgcc 38760 tcaacctcct gagtagctgg gattacagga acacaccacc atacccggctaatttttgga 38820 tttttggtag agacggggtt tcaccatgtt ggccaggctg gtctcgaactgacctcatga 38880 tctgcccgcc tcggcctccc aaagtgctag gattacaatg cttacacctagaacagatct 38940 gtcacctttc aaacttacag tgtgggcttg ttttgttatc aatgcattgatatttacagt 39000 acctatggat agtccatgta ctgaaataaa attgatttag gaattttgtcttataagtgt 39060 tctaaagact tgcacaagtg cacacataca cacactatat acatagtgtgtgtgcatgtg 39120 cgtgtatata aatgagtaac cttagactta gatttgttag atgaggaaggtttcaacctt 39180 ccccaaaatg caaatggaga atttcaacca tataaaccaa atattggcattttatctctg 39240 gaacacaaac atcttgtgtt actttatggt acttacgtaa tggcctgaatgctctagttt 39300 ttgccaatat attttacata attttgtata caagtttagt ggtatagaagataaaggaca 39360 ctaagcagga ttaacagctt ggttccctac agctgttaag tatgaaaacacaccatgaaa 39420 aggcaacaag cttcttccag gcaatggaag gctttttggg ggagaaaagaaagtgaatta 39480 caggtttaaa cctaggaatg tcattttttg aaacttgttt aaaatattttcaatccttct 39540 agtggtttgt gagctcctgg ggtttctgga aggtgtttgg gaactggatagagggttagt 39600 tcatgccttt aaaagccaat acatttccat ttctctttta taaccaagtaataacccaat 39660 tatgcatgta ttttatatac acagacacgt atttattttt actccaaaacaaaatggtct 39720 gaggcctttc aagaaagtgc atgtggcgaa gtcatggggg gcagggtggagaccatttgg 39780 tggtgcccac taactaggtt tctcagttgg cttatctctt agtggaccattgctagcaac 39840 cagggtgttt ttaagcattt gacagttttc catcactttt atttgccttcatatattgtt 39900 tcatttacac ccttagtatc tcttgtttta aagacaggag acaaaaagaacatggatatt 39960 taaatacaag ttaatgagga actttaaaat aataataatt ctacaaatttacctcaagat 40020 actttaccaa attcataagt tacatttatc tgatcaaaat tcttgtgtcacatatcaaga 40080 tgtttcttat acagcagaaa tcagtagaaa agaaaaaata ggccaagcgtgtggtggctc 40140 acacctgtaa tcccagtact ttgggaggcc aaggcaggag gattgcttgaggtttggagt 40200 tcaagaccag cctgggcaac acagtgagat cccatctcta ttaaaaaaattagaaaagaa 40260 aaagaataaa atggggctgt tatatccaaa ttggcttttt aaaaatcagcaataaggccg 40320 ggtgtggtgg ctcacacctg taattccagc actttggaag gctgaggcaggcggatcaat 40380 tgaggccaag agtttgagac cagcctggcg aacatggtga aaccctgtctgtactaaaaa 40440 tacaaaaatt agccaggcat gctggtgcat gcctgtaatc ccagttactcaggaggctga 40500 ggcaggagaa tcacttgaac ctgggaggtg gaggttgcag tgagctgagattgcaccact 40560 gcactccagc ctgagtgaca gagtgagacc ctgtctcaaa aaaaaagaaaaaaaaaattg 40620 gcaataaaaa caacctgttg cttgctggag gaaaaacctg cttgcaaagctcagtctgat 40680 atcatttttt aaacaaaact ctaagaacaa gccagtcagt taagctaaaaccaaatattt 40740 gattatgaaa agggtttttg tatattttta caggataaga tacaaataaatttcagtctt 40800 tcttttaata tgtatttctg ttcccaaacc agacacaaag caatttttaaacttgatcgt 40860 caagaaatct gttttctcct acacaatcaa tgaaaagtaa tctaaacagtgtttgtcagg 40920 ccaggcacag tggctcacat ctgtagtcct agcattttgg gaggcctaggcaggtagatt 40980 gcttgagccc agaatttcaa gaccagcctg gacaacatgg cgaaaccccatctgtattaa 41040 aaaaaaaaaa aaaaaaagac catatgtctg cagtcagatg gaaaaagtaaaaatatgtaa 41100 taaacacata tgaataatat taaggaccat attttaaaat aaacttgataataaattttt 41160 aataatatta tctacgataa aatgttttac ttaaatttcg ttctttatcatgccacacaa 41220 aaatggcaaa atgattaaga gagtttgcaa aattatgtgg tatagtgaaagaggtttgcg 41280 gttaaaaaaa aaaaagagag agagagagag aagtatgggg ccatggggatagtctctgta 41340 atcagtcacc tgaaccactt ttaatactca aaagacttat gagaataaaaatctgatttt 41400 tgctaagatt tattagcaaa ataaatctta ctccttcctg tccctctctaattatccttc 41460 agcttgacca tgtatgaaag aaaatttaca tttcactgtt taatctatttaaagatgaac 41520 atttcccatt aaatcaggat gcaccttata atcagtagca tctaacaatataagtcagcc 41580 aggctgcagt tgtgactgta gttagaattg cacatgtgtg aacatcaaatgagccagcat 41640 caaaacgtgc agaatggcca ggcacagtgg ctcacacctg tgatcccagcactttgggaa 41700 gctgaggtgg gtggatcact tgaggtcagg aattcaagac cagcctggccaagatggtga 41760 aatcacgttt ctactaaaaa tacaaaaatt agccaggcat ggtggcaggtgcctgtaatc 41820 ccagctactt ggtaggctaa gtcaggagaa tcgcttgaac ctgggaggcggaggttgcag 41880 tgagctgaga tcgcaccact gcactccagc ctgggcgaca gaccaagattccaccaaaaa 41940 aaaaaaaaaa attgcagaat tggtgtcagc gacttggaag aaaattctgcaaagaaaagt 42000 cctttttttt tctttttttt tttaaactcc taggaaccaa atggttgtggagaaggagta 42060 aatcagacat gtttagcaac attctttaag caggagtcaa aagtaagctaacactacata 42120 actgcaaggc cagcttagga gcccaggacc aatgactctc tgttgttttatggattattt 42180 taagaaatgc tgcatcatca aattcttaat atagaggatg atacatgggtaagtgtagac 42240 atcaaagagt ctgagtcaaa tgctgaatgt gaaaaagttt taggaataccgaaaccaatt 42300 tattttgctt aatgtttctc tttttcgtgt acaagtatgc tatatgagaaaataatctct 42360 atttaattaa atttataaca gccctttcaa taagtataaa atgaacattctgatcatgtc 42420 atagtttaac ttgcattttt ttgtcttaat ggcaaaaaac caatgacgcttcttacaatg 42480 atagcatctt agactcaatg aaaagtgggg atgaaatgaa atttggggatacagtacttt 42540 cccctcttct cctaaaacag ataatgagct tgaatgatct acaatgtttgctaactctac 42600 tgctttccta actgctgctc gtggtgttcc attttaataa aaagctgtgggctgttctta 42660 ttttgtttga catagggact ttttttttgg cccaagactt ttaatatcatgtggtccgta 42720 tttaactctc cctaaaatat ttcttgggaa gagaaattct agtagttcagtttcgcttgt 42780 atgatttctt tcaaagtgtc aatttactct tatttccttt gctaggggtgcaggctgcat 42840 ctttattgaa atgttccagg gtcaaccttt gtttcctggg gtttccaacatccttgaaca 42900 gctggagaaa atctgggagg taggagaata attcttctaa agaaaatgaaatatctgcat 42960 tttaagtttt gaaccaaatt tgccttacag acaaatgaag cagtccatctgctctgagat 43020 attaagccct atattaagat tgtagaaact gtagcatttg ccacagctataagcaccctg 43080 ggaatgtgtg gtcaggaaac tccctgttgc cccatagcag cccatgaatccagctcactg 43140 aatgatgttc aggtctcctg ctccctgtca ttagtattgt cttaacctcccagggcaatt 43200 tctgccatta ctactcagac atgtccctac cttgctacct ccagttctaatgctaccata 43260 tatttggccc tggatctttg tcaactgaaa ataagacata gaatttttagctgggtgcag 43320 tggctcatgc ctgtaatccc agcactttgg gattgctttg agcccaggagttcgagacca 43380 gcctgggcaa catggcgaaa ccccatccct acaaaaacaa aaatgagtgggctgtgtggc 43440 gcacacctta gtcccagcta ttcaggaggc tgagatggga ggatcacttgagcccaggga 43500 agtcgaggct gctgttagct gtgaccacgc cactgcactc caggctggggaacaaaaaaa 43560 agacacaaaa ttttcataga accctgatag aacagaggct ttccctcttagtgtgaaaga 43620 agtgtaccat ttatcatgct tatccacagc caaattccta aagtgtcaaggtgcctttgt 43680 gtgtgtatgc agctccattt cttaattcat tatttatccc taccgcagttgcctatgata 43740 tgctttgttt ttatggccct tatatagtat tacagtcata ctatagtcatctgtatattt 43800 ccttttttgg tcatattttt attgtggtaa aatatacaaa acaaaatttaccgtcttaac 43860 cctccttaag tgtacagctt gtcagcatta aatacattca tatagttgcaccaccatcac 43920 cgccatccat ttccagaact tctctatcat ccctaaggga agctctggacccactgaaca 43980 ataactgccc atcttccctc cccacactcc cctagcccct agtaacctctaatctacttt 44040 ctgtctccat gaattggcct attctaggta cctcatataa gtggaatcatacaaatttgt 44100 ctttccgtat ctggcttatg tcacttagca tattttcaag gttcatccatgttgtagaat 44160 gtgtcaaggg gctttaaatc ggcggggtgc aggggggtac tttattacttgctatcctgg 44220 atcctgctgc ttgtcttctg gctaaaataa aatgtacttt gtgaaattaagacattttat 44280 agagattaat tactgacatt aaattttctt ctagaaacat gggggctattatgaaggaac 44340 atgggaaaaa ctgggaagca ttcacaactg aaaaaaaaaa atccaagccaaaagactttt 44400 tctaaaaact ttcttgcaag acagagcaat gctatcttca cattatgttattgggtgcta 44460 taacatcatc taagctggag acagcctact gtcatagctt tggagtccaaagacctgggt 44520 ttgaattcta accattttct agctaaatga acatgggcaa gttatgtagtccctctgaac 44580 tttcgtttcc ttgtctgtaa aatggcaaca atgataataa ggactttctaattctttatt 44640 gagaattcca taaaaacaaa tgcataacaa gctccatgca ccataaatgctcaatagatg 44700 cttgctttct tcctgtccca tacaaattgt tgtacagatg tttcaataacctaactgcta 44760 gcaagtatta cctgaaattt aacccgattg ttctcttctt tcacttagcagtattatttc 44820 ttgtccacaa tagaggaagc acaattgcag ttctgatgct gcaatgaccttttatacatt 44880 tgaagagttt ttcctggtca tttaatcagg aaacaacact tactcaccatatatgaggcg 44940 agtaactcta caagactcta caaggtcttg taagaagcta taagccaagggggaaaaaaa 45000 aaagaagaat aagaaaaaca catgatctgt attttcaagt gttgttcagtctaggtaggg 45060 cgatgggtga agtatacgta aatatatgtg aaacaaacat aaactatgtatatatgtaaa 45120 aggatgtatg tatagatagt taatataaat tgtaatactg aaataagatgtgctattagg 45180 atacttgaag agtagtttat ttgaaaagaa tataagtata tccttgtgtgccattagtat 45240 ttgaagagtt gtatataaac tgattttttt tctttttcct tttttttgagaaggagtctt 45300 gctctgtcac ccaggctgga gtgcagtggt gccatctcgg ctcactgcaagctccacctc 45360 cccagttcaa gcgattctcc tgcctcagcc tcctgactag ctggaattacaggtgcccgc 45420 caccacacct ggctaacttt tgtattttta gtagagacgg ggtttcaccatgttggtcag 45480 gctggtctca aactcctgac ctcgtgatcc acccgctttg gcctcccaaagtggtgggat 45540 tacaggcgtg agccaccgcg cccagcctca taaactgatt tttaaaatacaatatacagt 45600 taggcatagt tgtgtgtgcc tatagtccct actgcttggg aggctgaggcaggaggatcc 45660 tttgatccca ggagtttggg caacatagtg agacccccat ctctaataataataaatata 45720 aatttcaaat aacattttaa aatatgacat actatctttg aatgaccacacaatttaaaa 45780 agcaatcatt ttacggttct ttagtgttca gttagcacag cacttagaaatcatagaata 45840 aagtgagcaa gatgcttctc aaagcctgat cactctttag gactcacaatgggctaggta 45900 ctatgctgga aagagaaaaa ataataattt tctaacctgc ttgagacatagtggtataaa 45960 tgataacaca gctgctgaac gtgatgactt tctcactttg tccgcagagcaagaaactat 46020 agatgcagta acaaaactgc attcaatgaa catgggactg tagataacaaactaacttca 46080 tttctttggg tacatgccct gtattgggat tgctggatca tatggtagttccatttttaa 46140 tattttgagg aacctccata ccatcttcca taatggctgt gctatttgcatgcccaccat 46200 cagtgtgcaa atgctccctt tcctccacat tcttgccaac acctctttcatctttttgat 46260 aatagttatg aggcaatatc tcaccatggt cctagacttc atttgtctgatgactaatga 46320 tattgagcat tttttcatat atctcttggc catttgtagg tcatcttttgagaaatgtgt 46380 attgaggttc ttagtccatt cctgctacca taacaaaatc ccttagagtgggcattttat 46440 aaagaacaga attggcccgg ggcgcagtgg ctcatgcctg taatcccagcactttgggag 46500 gccaaggtgg gtggatcacc tgaggtcagg agttcaagac cagcctggtcaatatggtga 46560 aaccccatct ctactaaaaa tacaaaaact agccgaacgt ggtggtgtgcacctgtagtc 46620 ccagctactt gggaggctga gacaggagaa ttgcttgaac ccaggaggaggaggttgcag 46680 tgagacgaga tcgtgccact gcactccagc ctgagcaaca gagtgagacttcatctcaaa 46740 aaaaaaaaaa aaaaaaaaaa aaagaacaga aatttatttc tcactgttctagaggctgga 46800 aagtccaaga tcaaggcact gtaggctgtt gtccagtgag tatatttggtctccaagtta 46860 gtgccttgtc gctgcatcct ccagataggg caaatgctgt gtccttacatggtggaaggg 46920 tagaagagca aacgggcctg actgattccc tctagctcct ttataagggcattcatctct 46980 gtccttgtgt cctaatcaca cgctaaaggt ggctaaaggc cccacctcttaatactgttg 47040 cattggggat aaagtttcaa catgaattat gaagagaata caaacatttaaaccacaaca 47100 agtcctttgc ccactttttt tttggagacc gagtctcact ctgttgcccaggctggaatg 47160 cagtggcttg atcctggctc attgcaacct ccacctcctg ggttcaagcaattctcctgc 47220 ctcagcttcc caagtagctg ggattacagg tgtgcactac cacacccagctaattttgta 47280 tatttagtag agacagggtt ttaccatgtt agccaggctg atctcgaactctcgacttct 47340 ggtgatccac ctgcctcagc ctcccaaagt gctgagatta caggcgtgagccaccgtgcc 47400 cggccctttg cccactgttt aatggggttg tcttcttgct attgagttccttatatattt 47460 tttatattaa ccccttatca aatgtatggc ttgcaaatat tttctcccatcgtaggttgt 47520 ctcttcactc taatgattgt ttcctttgct ctgaagacac tttttagttttatttattcc 47580 catttgtcta ttttcacatt tgttgcctat aagcaggtta gaaaattatacagattataa 47640 atagttcctg aatttgtgtt ttactaaacg tagcctacac agatgaaaacaggaaagcta 47700 cacttcagaa tctgtgatat ttgatgtcag aagtgcatcc ctgaaagcaatgggtccatt 47760 ctaaatctcc taacctctaa ccataatttg ttctatattt atcctgagatctcactctta 47820 ggaataaaaa cacattgaga agtcctgagt ctctatttta ctatttttctgaagtgcctg 47880 tagtgtgtgt gtttacatct aaataatagc tgtcaccact ttctgatcaattttaaaaac 47940 taattttaaa taagtgtttt tcataaataa tcctggattt agttctaaaatcagaataaa 48000 ctatgcaaac tttgaatcca ttaatcaaaa tgcttttagt ttccattccaacaaaggcag 48060 ataaacagcc ccttcagacc actgtggttt gaaacatagc actcactggctgccttttaa 48120 gagccttcag ggagggagca aaacaacaat ttttggtttt cagtttcccagacagtgaag 48180 gagagattta gtaattttct caagtgaaaa agaattcaat aacttgcaaatagaaactga 48240 gatcaaattt ccaaataaag tatattgaat ttttgtttaa acttttaaaatctcaagctt 48300 aaagctttga acataagatt aaaaaaactt tttttagtat ccattttgttggctttagtt 48360 aaatatcata caaagtaacc aaccatctgg taactttcac cttagagaaaacatgatagt 48420 ggttgtcacc tatttcttct attgttttct cttcattatc tttgctttcttttcactgca 48480 ctttgccagc caacagagga tgtatgggta catgtgactc acacccacttgtttacacat 48540 gcatctgtgc aaatacataa gatggtaggt taaaaaaaga agaattagtttcttgtcccc 48600 tggccttctc ccacaaaaga agaattagtc cagttggttt ttcaaaatggattccaggat 48660 tcttagtgtt ccctcgggct cagggtggtt gataggaaaa gcctataatcctctcagtca 48720 cttttcagtt tgtttaggga atggatcaaa gaaggaagat tttactgggtggcatgattt 48780 ttttattata tgagggaaaa tagcacttca ctgtcttttg tttaaagacaagcttaacag 48840 atgctaaaaa gtacatctct cagccagatt cctagtcaac aagctgatagacactaagat 48900 tctggattct tcattgatta tattcagtca ttgttgggca attgactccctgccataata 48960 attgggccag tatctataac cagcatttta cagatggatt cgctagactctttctgtaag 49020 agatgtttct aaaaagagtt atagtgagat atgcttctaa gaaaagttatactgtagtag 49080 tgtaatgaaa gctactagtg ttttattagt atttcacaag aacaatgttactctgtctcc 49140 catatataac tgtctatggg cttttatgat tattctttaa aaaaaaaaaatactaaggta 49200 atgcctaccg gggaactcat ggtgctggct tcatccaaag tctgagctgttttggcttta 49260 tactccgaaa gactttattt tcatacatct taactaaaaa ctggggctttaaattggtca 49320 ttcaaggcca ggcgcggttg ctcatgcctg aaatcccagc actttgggaggccgaggtgg 49380 gcagatcacg aggtcaggag attgagacca tcctggccaa cacggtgaaaccccgtctct 49440 actaaaaata caacaacaac aacaacaaaa atagccaggc gtggtggcttgcatctgtaa 49500 tcccagctac tcaggaggct gaggcaggag aatggtgtga acctgggaggcagagcttgc 49560 agtgagccga gatcgcatca ctgcactcca gcctgggcga cagagcgagactccgtctca 49620 aaaaaaaaaa acatcggtaa ttcaaagcat agaccagccc tttttcaagtgatgttgttc 49680 ccatgacaat ccatcagtga aaaaccaaat accatattcc aagctgctagtcacagagaa 49740 aacaagcaga tgagatgaat gtaatagaaa agactagagt tagttttggggtcatcttta 49800 gccaacattc cattgcctga agctcagtaa tctgaatcct ttttaatttgagcacatcag 49860 ggaacagctg aatacccatg ctgaggcata atttaagctg tcaagtgtctcctgtcaata 49920 tacatgtggt catctgatgc aaggcaaaga gacagtcact cctgcttctttatatcccta 49980 gctcccaaca tggtgtccta atgcatgata atcatgcagt aaatgttcagtgatgagaac 50040 atgactttga gcaaggctgt atgatctgcc tcagaacaag tgagtcagtaagaatgcagg 50100 ccccggacca taggaatgta ttacagtttt gcccaagaaa ccacaaacgttggaaacact 50160 caagtttctt tctcgtatac atcagctggt gtcatgcaat gggacataccatctgacgct 50220 tccctgttct tccctgattt gtcctgcatg tctccaatac ctctttccaaccacctcatc 50280 tccccacctc acctttcttt ttctttgttt ggctttatat aggtgctgggagtccctaca 50340 gaggatactt ggccgggagt ctccaagcta cctaactaca atccaggtaatattgatctg 50400 agcttctgaa tactctgaga attagtaatg taaggagagc attggccacgctaacagggc 50460 gttcttgtat tgtgaactca gcggcaaaga tgggtgtaga ggaatttctacattcatata 50520 ttccctgact aatctttgta tgaggaagac actgaaagag tagctgaggttagaccagtt 50580 ccccagctct gtaaaacaca agtagcaagc tgaatagaat ttgaaatgactattactgtg 50640 gattccacat ccattgtcaa atacccaatg gctcaaaaga acaactcaaaagatgggctc 50700 acttttgggc cccctgactg tcataagtgt attgattagt attgaattgcatatgtataa 50760 aaagaaagct aatgcaacag aacagaggta gaggctcgct aggcctaggacatgccaagt 50820 aagctgtctg taggttatac ttactaagag ttcattcatt gcctgtaaacctgacacttg 50880 gtcattgtct ctcacacatt tcatctttca agactggctt ctgggatcgatttagaagtg 50940 ctggaagtgt tatccatggg ggaattcttt gagaagctgt cgcagggccacatcagaggg 51000 atcagattaa gcagtagtca cttcaaggat gttgagacag aggggaggagacaggcactg 51060 aactacagga tgaaggatca tattagaagc tgaagaagca aataaagcccatgccaaagc 51120 tgagctctca ctggcagggt tgaaggggag gtagaaaggt acagataacgacaagattag 51180 ggtggatatg ctccaagcca gatttttcta gtctttatgg tcttacattgttccattact 51240 aaaaatgaaa tcttcccaaa ttgttgtcct tacttttttt tttttttttttgagatggag 51300 ttttgctctt atcgcccagg ctggagtgca gtggcacgat ctcggctcactgcaacctcc 51360 acctcctggg ttcaagcaat tctcctgcct cagcctcccc aagtagctgggactacaggc 51420 acccgccacc atgcccagct aattttttgt atttttagta gagatgaggtttcaccatgt 51480 tggccaggct ggtctcgaac tcctgacctc aggtgatcca cttgcttcagcttcccaaaa 51540 tgctgggatt acaggcatga gccagcgcgc ctggcctgtt gtccttactaactttggtat 51600 gagattatcc tggaagggtt tcctgagagc aagaaattgt aggtagagttaaaatgtgat 51660 taaagaagag aataaaatac atagggagct ggggactctt tttcttattttctttagcat 51720 ccaatacttt tgcttacagc tatccatagg gatctggcat cttgaaccaccaggattatg 51780 gaagccctac agcaagctaa agactaactg taaagtcctt tcagctgctttgtgaatggt 51840 tatatctatt gctaaaaggc cttaatatca tttgcaaata gtttatgatttctaactatt 51900 tttctagagt ttaacacgtg agaaaaatgc tactctctgg tcacaggacttagaatagtg 51960 cctatttcca ttggtctgag atagagaaaa aagaacaagt ttcttgtggagccgtggtcc 52020 agtctgcaaa ttgctcctat ctccagttgc catggtttcc aggagaacgtggctctcatc 52080 ttttcctgcc ctgcctgtac ttctccctgt ccactctgtt ctctattttccctcagcttc 52140 ctaactgagg atgccagcag aagtttagag tcacagatgg attgtaggaaacaatttgga 52200 tgatgccaat acaaagctac tgtggtgggc atatgctgct cccccaaacttcagacattt 52260 gggtttcagg ttggtccagg caatcaacag tgatccttaa tacaaaatgtcttggtgaga 52320 gcaataatca agaaacttgg ccaaagtgct tccctgccag attgtgtgcttaataagata 52380 actgggttcc aataaaacag agaaaatatg ttacatttta aaaaattttctgttgtttca 52440 aaacaatgtg cagtttttct atataagaag aaaagtctcc aggcccaacatccatagggc 52500 tcatcatcca ttgtttttct tttaagtttt caatttaatc caaataagtcaaaaattttc 52560 aggtacctac tatctgccag gtgctgtgcc gtgcgctggg gctacacagatggagagggt 52620 gcattcttgg atctctagtg tttgggtttg gattcattca cccacactctttcaccagtt 52680 ctctttgtta ctggggtgct catttgtgag ccctgcttcc atggcttggagagtttgtgg 52740 ctgtgggcca ggctgagctt atggagcaaa gggagttgga accttagccatagacatgat 52800 gtctaaacct ggatttggaa atcttaaaag tccagcctat cttgggccatggggtcagta 52860 ttattgataa ctcaatccca aggactgtgt tttaaaaggg tctccaacatctgcatttca 52920 ggaacatcct cttacgtgag tcaataagtt ccttttgagc caccccctacccatccccat 52980 ccctgagctg ctgtggcttc taaacacttg aatgtcagtg attaaggggagcagaagaca 53040 agctgggagc caggaaagtg tcacagatga gcaccgtgtc agcagcattctggatgagct 53100 tcccattcct ttccttttca ttctaagtag tcctaggagc ccccaaactttgaatcagcc 53160 agtacaattt tgagggagtc cagttgtccg gaacttggga gaaccatccagtgtccatct 53220 acacccatgc ctccatttct aggccttatc tggacacctc taggaggacagcaaagtttc 53280 catttgtaca gcttttaaaa agtcacctga tgctgaccca gtcggatttctc 53332 4 1308 DNA Human 4 atgggtcaag agctgtgtgc aaagactgta cagcctggatgcagctgcta ccattgttca 60 gagggaggcg aggcacacag ctgtcggagg agtcagcctgagaccacgga ggctgcgttc 120 aagctaacag acctaaaaga agcatcatgt tccatgacttcatttcaccc caggggactt 180 caagctgccc gtgcccagaa gttcaagagt aaaaggccacggagtaacag tgattgtttt 240 caggaagagg atctgaggca gggttttcag tggaggaagagcctcccttt tggggcagcc 300 tcatcttact tgaacttgga gaagctgggt gaaggctcttatgcgacagt ttacaagggg 360 attagcagaa taaatggaca actagtggct ttaaaagtcatcagcatgaa tgcagaggaa 420 ggagtcccat ttacagctat ccgagaagct tctctcctgaagggtttgaa acatgccaat 480 attgtgctcc tgcatgacat aatccacacc aaagagacactgacattcgt ttttgaatac 540 atgcacacag acctggccca gtatatgtct cagcatccaggagggcttca tcctcataat 600 gtcagacttt tcatgtttca acttttgcgg ggcctggcgtacatccacca ccaacacgtt 660 cttcacaggg acctgaaacc tcagaactta ctcatcagtcacctgggaga gctcaaactg 720 gctgattttg gtcttgcccg ggccaagtcc attcccagccagacatactc ttcagaagtc 780 gtgaccctct ggtaccggcc ccctgatgct ttgctgggagccactgaata ttcctctgag 840 ctggacatat ggggtgcagg ctgcatcttt attgaaatgttccagggtca acctttgttt 900 cctggggttt ccaacatcct tgaacagctg gagaaaatctgggaggtgct gggagtccct 960 acagaggata cttggccggg agtctccaag ctacctaactacaatccaga atggttccca 1020 ctgcctacgc ctcgaagcct tcatgttgtc tggaacaggctgggcagggt tcctgaagct 1080 gaagacctgg cctcccagat gctaaaaggc tttcccagagaccgcgtctc cgcccaggaa 1140 gcacttgttc atgattattt cagcgccctg ccatctcagctgtaccagct tcctgatgag 1200 gagtctttgt ttacagtttc aggagtgagg ctaaagccagaaatgtgtga ccttttggcc 1260 tcctaccaga aaggtcacca cccagcccag tttagcaaatgctggtga 1308 5 435 PRT Human 5 Met Gly Gln Glu Leu Cys Ala Lys Thr ValGln Pro Gly Cys Ser Cys 1 5 10 15 Tyr His Cys Ser Glu Gly Gly Glu AlaHis Ser Cys Arg Arg Ser Gln 20 25 30 Pro Glu Thr Thr Glu Ala Ala Phe LysLeu Thr Asp Leu Lys Glu Ala 35 40 45 Ser Cys Ser Met Thr Ser Phe His ProArg Gly Leu Gln Ala Ala Arg 50 55 60 Ala Gln Lys Phe Lys Ser Lys Arg ProArg Ser Asn Ser Asp Cys Phe 65 70 75 80 Gln Glu Glu Asp Leu Arg Gln GlyPhe Gln Trp Arg Lys Ser Leu Pro 85 90 95 Phe Gly Ala Ala Ser Ser Tyr LeuAsn Leu Glu Lys Leu Gly Glu Gly 100 105 110 Ser Tyr Ala Thr Val Tyr LysGly Ile Ser Arg Ile Asn Gly Gln Leu 115 120 125 Val Ala Leu Lys Val IleSer Met Asn Ala Glu Glu Gly Val Pro Phe 130 135 140 Thr Ala Ile Arg GluAla Ser Leu Leu Lys Gly Leu Lys His Ala Asn 145 150 155 160 Ile Val LeuLeu His Asp Ile Ile His Thr Lys Glu Thr Leu Thr Phe 165 170 175 Val PheGlu Tyr Met His Thr Asp Leu Ala Gln Tyr Met Ser Gln His 180 185 190 ProGly Gly Leu His Pro His Asn Val Arg Leu Phe Met Phe Gln Leu 195 200 205Leu Arg Gly Leu Ala Tyr Ile His His Gln His Val Leu His Arg Asp 210 215220 Leu Lys Pro Gln Asn Leu Leu Ile Ser His Leu Gly Glu Leu Lys Leu 225230 235 240 Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Ile Pro Ser Gln ThrTyr 245 250 255 Ser Ser Glu Val Val Thr Leu Trp Tyr Arg Pro Pro Asp AlaLeu Leu 260 265 270 Gly Ala Thr Glu Tyr Ser Ser Glu Leu Asp Ile Trp GlyAla Gly Cys 275 280 285 Ile Phe Ile Glu Met Phe Gln Gly Gln Pro Leu PhePro Gly Val Ser 290 295 300 Asn Ile Leu Glu Gln Leu Glu Lys Ile Trp GluVal Leu Gly Val Pro 305 310 315 320 Thr Glu Asp Thr Trp Pro Gly Val SerLys Leu Pro Asn Tyr Asn Pro 325 330 335 Glu Trp Phe Pro Leu Pro Thr ProArg Ser Leu His Val Val Trp Asn 340 345 350 Arg Leu Gly Arg Val Pro GluAla Glu Asp Leu Ala Ser Gln Met Leu 355 360 365 Lys Gly Phe Pro Arg AspArg Val Ser Ala Gln Glu Ala Leu Val His 370 375 380 Asp Tyr Phe Ser AlaLeu Pro Ser Gln Leu Tyr Gln Leu Pro Asp Glu 385 390 395 400 Glu Ser LeuPhe Thr Val Ser Gly Val Arg Leu Lys Pro Glu Met Cys 405 410 415 Asp LeuLeu Ala Ser Tyr Gln Lys Gly His His Pro Ala Gln Phe Ser 420 425 430 LysCys Trp 435 6 240 PRT Mus musculus 6 Phe Gly Lys Ala Asp Ser Tyr Glu LysLeu Glu Lys Leu Gly Glu Gly 1 5 10 15 Ser Tyr Ala Thr Val Tyr Lys GlyLys Ser Lys Val Asn Gly Lys Leu 20 25 30 Val Ala Leu Lys Val Ile Arg LeuGln Glu Glu Glu Gly Thr Pro Phe 35 40 45 Thr Ala Ile Arg Glu Ala Ser LeuLeu Lys Gly Leu Lys His Ala Asn 50 55 60 Ile Val Leu Leu His Asp Ile IleHis Thr Lys Glu Thr Leu Thr Leu 65 70 75 80 Val Phe Glu Tyr Val His ThrAsp Leu Cys Gln Tyr Met Glu Gln His 85 90 95 Pro Gly Gly Leu His Pro AspAsn Val Lys Leu Phe Leu Phe Gln Leu 100 105 110 Leu Arg Gly Leu Ser TyrIle His Gln Arg Tyr Ile Leu His Arg Asp 115 120 125 Leu Lys Pro Gln AsnLeu Leu Ile Ser Asp Thr Gly Glu Leu Lys Leu 130 135 140 Ala Asp Phe GlyLeu Ala Arg Ala Lys Ser Val Pro Ser His Thr Tyr 145 150 155 160 Ser AsnGlu Val Val Thr Leu Trp Tyr Arg Pro Pro Asp Val Leu Leu 165 170 175 GlySer Thr Glu Tyr Ser Thr Cys Leu Asp Met Trp Gly Val Gly Cys 180 185 190Ile Phe Val Glu Met Ile Gln Gly Val Ala Ala Phe Pro Gly Met Lys 195 200205 Asp Ile Gln Asp Gln Leu Glu Arg Ile Phe Leu Val Leu Gly Thr Pro 210215 220 Asn Glu Asp Thr Trp Pro Gly Val His Ser Leu Pro His Phe Lys Pro225 230 235 240 7 245 PRT Human 7 Phe Gly Lys Ala Asp Ser Tyr Glu LysLeu Glu Lys Leu Gly Glu Gly 1 5 10 15 Ser Tyr Ala Thr Val Tyr Lys GlyLys Ser Lys Val Asn Gly Lys Leu 20 25 30 Val Ala Leu Lys Val Ile Arg LeuGln Glu Glu Glu Gly Thr Pro Phe 35 40 45 Thr Ala Ile Arg Glu Ala Ser LeuLeu Lys Gly Leu Lys His Ala Asn 50 55 60 Ile Val Leu Leu His Asp Ile IleHis Thr Lys Glu Thr Leu Thr Leu 65 70 75 80 Val Phe Glu Tyr Val His ThrAsp Leu Cys Gln Tyr Met Asp Lys His 85 90 95 Pro Gly Gly Leu His Pro AspAsn Val Lys Leu Phe Leu Phe Gln Leu 100 105 110 Leu Arg Gly Leu Ser TyrIle His Gln Arg Tyr Ile Leu His Arg Asp 115 120 125 Leu Lys Pro Gln AsnLeu Leu Ile Ser Asp Thr Gly Glu Leu Lys Leu 130 135 140 Ala Asp Phe GlyLeu Ala Arg Ala Lys Ser Val Pro Ser His Thr Tyr 145 150 155 160 Ser AsnGlu Val Val Thr Leu Trp Tyr Arg Pro Pro Asp Val Leu Leu 165 170 175 GlySer Thr Glu Tyr Ser Thr Cys Leu Asp Met Trp Gly Val Gly Cys 180 185 190Ile Phe Val Glu Met Ile Gln Gly Val Ala Ala Phe Pro Gly Met Lys 195 200205 Asp Ile Gln Asp Gln Leu Glu Arg Ile Phe Leu Val Leu Gly Thr Pro 210215 220 Asn Glu Asp Thr Trp Pro Gly Val His Ser Leu Pro His Phe Lys Pro225 230 235 240 Glu Arg Phe Thr Leu 245 8 330 PRT Mus musculus 8 Phe GlyLys Ala Asp Ser Tyr Glu Lys Leu Glu Lys Leu Gly Glu Gly 1 5 10 15 SerTyr Ala Thr Val Tyr Lys Gly Lys Ser Lys Val Asn Gly Lys Leu 20 25 30 ValAla Leu Lys Val Ile Arg Leu Gln Glu Glu Glu Gly Thr Pro Phe 35 40 45 ThrAla Ile Arg Glu Ala Ser Leu Leu Lys Gly Leu Lys His Ala Asn 50 55 60 IleVal Leu Leu His Asp Ile Ile His Thr Lys Glu Thr Leu Thr Leu 65 70 75 80Val Phe Glu Tyr Val His Thr Asp Leu Cys Gln Tyr Met Glu Gln His 85 90 95Pro Gly Gly Leu His Pro Asp Asn Val Lys Leu Phe Leu Phe Gln Leu 100 105110 Leu Arg Gly Leu Ser Tyr Ile His Gln Arg Tyr Ile Leu His Arg Asp 115120 125 Leu Lys Pro Gln Asn Leu Leu Ile Ser Asp Thr Gly Glu Leu Lys Leu130 135 140 Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Val Pro Ser His ThrTyr 145 150 155 160 Ser Asn Glu Val Val Thr Leu Trp Tyr Arg Pro Pro AspVal Leu Leu 165 170 175 Gly Ser Thr Glu Tyr Ser Thr Cys Leu Asp Met TrpGly Val Gly Cys 180 185 190 Ile Phe Val Glu Met Ile Gln Gly Val Ala AlaPhe Pro Gly Met Lys 195 200 205 Asp Ile Gln Asp Gln Leu Glu Arg Ile PheLeu Val Leu Gly Thr Pro 210 215 220 Asn Glu Asp Thr Trp Pro Gly Val HisSer Leu Pro His Phe Lys Pro 225 230 235 240 Glu Arg Phe Thr Val Tyr SerSer Lys Ser Leu Arg Gln Ala Trp Asn 245 250 255 Lys Leu Ser Tyr Val AsnHis Ala Glu Asp Leu Ala Ser Lys Leu Leu 260 265 270 Gln Cys Ser Pro LysAsn Arg Leu Ser Ala Gln Ala Ala Leu Ser His 275 280 285 Glu Tyr Phe SerAsp Leu Pro Pro Arg Leu Trp Glu Leu Thr Asp Met 290 295 300 Ser Ser IlePhe Thr Val Pro Asn Val Arg Leu Gln Pro Glu Ala Gly 305 310 315 320 GluSer Met Arg Ala Phe Gly Lys Asn Asn 325 330 9 330 PRT Human 9 Phe GlyLys Ala Asp Ser Tyr Glu Lys Leu Glu Lys Leu Gly Glu Gly 1 5 10 15 SerTyr Ala Thr Val Tyr Lys Gly Lys Ser Lys Val Asn Gly Lys Leu 20 25 30 ValAla Leu Lys Val Ile Arg Leu Gln Glu Glu Glu Gly Thr Pro Phe 35 40 45 ThrAla Ile Arg Glu Ala Ser Leu Leu Lys Gly Leu Lys His Ala Asn 50 55 60 IleVal Leu Leu His Asp Ile Ile His Thr Lys Glu Thr Leu Thr Leu 65 70 75 80Val Phe Glu Tyr Val His Thr Asp Leu Cys Gln Tyr Met Asp Lys His 85 90 95Pro Gly Gly Leu His Pro Asp Asn Val Lys Leu Phe Leu Phe Gln Leu 100 105110 Leu Arg Gly Leu Ser Tyr Ile His Gln Arg Tyr Ile Leu His Arg Asp 115120 125 Leu Lys Pro Gln Asn Leu Leu Ile Ser Asp Thr Gly Glu Leu Lys Leu130 135 140 Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Val Pro Ser His ThrTyr 145 150 155 160 Ser Asn Glu Val Val Thr Leu Trp Tyr Arg Pro Pro AspVal Leu Leu 165 170 175 Gly Ser Thr Glu Tyr Ser Thr Cys Leu Asp Met TrpGly Val Gly Cys 180 185 190 Ile Phe Val Glu Met Ile Gln Gly Val Ala AlaPhe Pro Gly Met Lys 195 200 205 Asp Ile Gln Asp Gln Leu Glu Arg Ile PheLeu Val Leu Gly Thr Pro 210 215 220 Asn Glu Asp Thr Trp Pro Gly Val HisSer Leu Pro His Phe Lys Pro 225 230 235 240 Glu Arg Phe Thr Leu Tyr SerSer Lys Asn Leu Arg Gln Ala Trp Asn 245 250 255 Lys Leu Ser Tyr Val AsnHis Ala Glu Asp Leu Ala Ser Lys Leu Leu 260 265 270 Gln Cys Ser Pro LysAsn Arg Leu Ser Ala Gln Ala Ala Leu Ser His 275 280 285 Glu Tyr Phe SerAsp Leu Pro Pro Arg Leu Trp Glu Leu Thr Asp Met 290 295 300 Ser Ser IlePhe Thr Val Pro Asn Val Arg Leu Gln Pro Glu Ala Gly 305 310 315 320 GluSer Met Arg Ala Phe Gly Lys Asn Asn 325 330 10 330 PRT Mus musculus 10Phe Gly Lys Ala Asp Ser Tyr Glu Lys Leu Glu Lys Leu Gly Glu Gly 1 5 1015 Ser Tyr Ala Thr Val Tyr Lys Gly Lys Ser Lys Val Asn Gly Lys Leu 20 2530 Val Ala Leu Lys Val Ile Arg Leu Gln Glu Glu Glu Gly Thr Pro Phe 35 4045 Thr Ala Ile Arg Glu Ala Ser Leu Leu Lys Gly Leu Lys His Ala Asn 50 5560 Ile Val Leu Leu His Asp Ile Ile His Thr Lys Glu Thr Leu Thr Leu 65 7075 80 Val Phe Glu Tyr Val His Thr Asp Leu Cys Gln Tyr Met Asp Lys His 8590 95 Pro Gly Gly Leu His Pro Asp Asn Val Lys Leu Phe Leu Phe Gln Leu100 105 110 Leu Arg Gly Leu Ser Tyr Ile His Gln Arg Tyr Ile Leu His ArgAsp 115 120 125 Leu Lys Pro Gln Asn Leu Leu Ile Ser Asp Thr Gly Glu LeuLys Leu 130 135 140 Ala Asp Phe Gly Leu Ala Arg Ala Lys Ser Val Pro SerHis Thr Tyr 145 150 155 160 Ser Asn Glu Val Val Thr Leu Trp Tyr Arg ProPro Asp Val Leu Leu 165 170 175 Gly Ser Thr Glu Tyr Ser Thr Cys Leu AspMet Trp Gly Val Gly Cys 180 185 190 Ile Phe Val Glu Met Ile Gln Gly ValAla Ala Phe Pro Gly Met Lys 195 200 205 Asp Ile Gln Asp Gln Leu Glu ArgIle Phe Leu Val Leu Gly Thr Pro 210 215 220 Asn Glu Asp Thr Trp Pro GlyVal His Ser Leu Pro His Phe Lys Pro 225 230 235 240 Glu Arg Phe Thr ValTyr Asn Ser Lys Ser Leu Arg Gln Ala Trp Asn 245 250 255 Lys Leu Ser TyrVal Asn His Ala Glu Asp Leu Ala Ser Lys Leu Leu 260 265 270 Gln Cys SerPro Lys Asn Arg Leu Ser Ala Gln Ala Ala Leu Ser His 275 280 285 Glu TyrPhe Ser Asp Leu Pro Pro Arg Leu Trp Glu Leu Thr Asp Met 290 295 300 SerSer Ile Phe Thr Val Pro Asn Val Arg Leu Gln Pro Glu Ala Gly 305 310 315320 Glu Ser Met Arg Ala Phe Gly Lys Asn Asn 325 330

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence selected from the group consisting of SEQ ID NOS: 2 and 5; (b)an amino acid sequence of an allelic variant of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2 and 5, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule selected from the group consisting of SEQ ID NOS: 1, 3, and 4;(c) an amino acid sequence of an ortholog of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2 and 5, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeselected from the group consisting of SEQ ID NOS: 1, 3, and 4; and (d) afragment of an amino acid sequence selected from the group consisting ofSEQ ID NOS: 2 and 5, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence selected from the group consisting of SEQ ID NOS: 2 and 5; (b)an amino acid sequence of an allelic variant of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2 and 5, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule selected from the group consisting of SEQ ID NOS: 1, 3, and 4;(c) an amino acid sequence of an ortholog of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2 and 5, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeselected from the group consisting of SEQ ID NOS: 1, 3, and 4; and (d) afragment of an amino acid sequence selected from the group consisting ofSEQ ID NOS: 2 and 5, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 2 and 5; (b) a nucleotide sequencethat encodes of an allelic variant of an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 2 and 5, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule selected from the groupconsisting of SEQ ID NOS: 1, 3, and 4; (c) a nucleotide sequence thatencodes an ortholog of an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 2 and 5, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule selected from the group consisting of SEQ ID NOS:1, 3, and 4; (d) a nucleotide sequence that encodes a fragment of anamino acid sequence selected from the group consisting of SEQ ID NOS: 2and 5, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence selectedfrom the group consisting of SEQ ID NOS: 2 and 5; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2 and 5, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule selected from the groupconsisting of SEQ ID NOS: 1, 3, and 4; (c) a nucleotide sequence thatencodes an ortholog of an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 2 and 5, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule selected from the group consisting of SEQ ID NOS:1, 3, and 4; (d) a nucleotide sequence that encodes a fragment of anamino acid sequence selected from the group consisting of SEQ ID NOS: 2and 5, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human kinase protein, said methodcomprising administering to a patient a pharmaceutically effectiveamount of an agent identified by the method of claim
 16. 19. A methodfor identifying a modulator of the expression of a peptide of claim 2,said method comprising contacting a cell expressing said peptide with anagent, and determining if said agent has modulated the expression ofsaid peptide.
 20. An isolated human kinase peptide having an amino acidsequence that shares at least 70% homology with an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2 and
 5. 21. A peptideaccording to claim 20 that shares at least 90 percent homology with anamino acid sequence selected from the group consisting of SEQ ID NOS: 2and
 5. 22. An isolated nucleic acid molecule encoding a human kinasepeptide, said nucleic acid molecule sharing at least 80 percent homologywith a nucleic acid molecule selected from the group consisting of SEQID NOS: 1, 3, and
 4. 23. A nucleic acid molecule according to claim 22that shares at least 90 percent homology with a nucleic acid moleculeselected from the group consisting of SEQ ID NOS: 1, 3, and 4.